CN1882702A - Synthetic lethal screen using RNA interference - Google Patents
Synthetic lethal screen using RNA interference Download PDFInfo
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Abstract
The invention provides a method for identifying one or more genes in a cell of a cell type which interact with, e.g., modulate the effect of, an agent, e.g., a drug. For example, an identified gene may confer resistance or sensitivity to a drug, i.e., reduces or enhances the effect of the drug. The invention also provides STK6 and TPX2 as a gene that exhibits synthetic lethal interactions with KSP encoding a kinesin-like motor protein, and methods and compositions for treatment of diseases, e.g., cancers, by modulating the expression of STK6 or TPX2 gene and/or the activity of STK6 or TPX2 gene product. The invention also provides genes involved in cellular response to DNA damage, and their therapeutic uses.
Description
The application requires the U.S. Provisional Patent Application No.60/554 of submission on March 17th, 2004 according to 35 U.S.C.119 (e), 284, the U.S. Provisional Patent Application No.60/548 that submitted on February 27th, 2004,568, with the U.S. Provisional Patent Application No.60/505 that submitted on September 22nd, 2003,229 rights and interests, described application is incorporated herein by reference in full at this.
1. invention field
The present invention relates to disturb the screening that interacts, for example, cause death/collaborative (synthetic lethal) method for screening and the composition that cause death with RNA.The invention still further relates to and KSP, promptly kinesin sample dynein has the collaborative interactional gene that causes death, and therepic use.The invention still further relates to the gene of participation to DNA destructive cell response, and therepic use.
2. background of invention
It is the genetic expression that is used for preventing mammalian cell that RNA disturbs (RNAi), and produces effective ways (Couzin, 2002, the Science 298:2296-2297 of a lot of vibrations in scientific domain; McManus et al., 2002, Nat.Rev.Genet.3,737-747; Hannon, G.J., 2002, Nature 418,244-251; Paddison et al., 2002, Cancer Cell 2,17-23).RNA disturbs and guards in the evolutionary process from the caenorhabditis elegant to people, and is considered to not be subjected to work in the RNA viruses intrusion at the protection cell.When cell was subjected to the dsRNA virus infection, it is fixed that dsRNA is identified with target, so that cut by the III type RNA enzyme that is called " nickase (Dicer) ".The nickase enzyme becomes the duplex of short 21nt with RNA " stripping and slicing ", is called siRNA or short RNA interfering, and it comprises the complete paired ribonucleotide of 19nt and two unpaired Nucleotide on every chain 3 ' end.These short duplexs associate with the polyprotein mixture that is called RISC, and these mixtures are navigated to the mRNA transcript that has sequence similarity with siRNA.Therefore, be present in the nuclease cutting mRNA transcript in the RISC mixture, thus the expression of cancellation gene product.Under the situation of virus infection, this mechanism will cause the destruction of virus transcription thing, stop virus synthetic thus.Because siRNAs is double-stranded, the potential that arbitrary chain all has with RISC associates and guidance has the transcript silence of sequence similarity.
The specific gene silence provides utilizes human genome data interpretation gene function, identifies the potential of the treatment that medicine target and exploitation are more special.A lot of such siRNA specificitys that height is provided as their target of target that are applied as.With the hybridization that contains with the transcript of the partial identity of siRNA sequence, it is exophytic not as the phenotype of the silence of the transcript of target to induce reflection to remove target gene.This can make identifies that the gene with phenotypic correlation becomes chaotic.Many reports in the document have been pointed out the accurate specificity of siRNA, pointed out to the requirement of identity (Elbashir etal., the 2001.EMBOJ.20:6877-6888 completely that be close to of siRNA sequence; Tuschl et al., 1999, Genes Dev.13:3191-3197; Hutvagneret al., Sciencexpress 297:2056-2060).A up-to-date report shows, the sequence complementarity is that the fixed transcript cutting of siRNA target is necessary completely, to under the situation of not carrying out the transcript degraded, the mode with microRNA cause translation repression (Hutvagner et al., Sciencexpress 297:2056-2060) and part is complementary.
To little adjusting RNA, comprise that the function of siRNA and miRNA does not also fully understand.A ubiquitous problem relates to the mechanism of the different reticent approach of determining this two classes adjusting RNA.MiRNA is the adjusting RNA by genomic expression, and from the processing of precursor stem-ring structure, is incorporated into single-chain nucleic acid (Leeet al., 1993, the Cell 75:843-854 of the sequence among 3 ' UTR of said target mrna with generation; Reinhart et al., 2000, Nature 403:901-906; Lee et al., 2001, Science 294:862-864; Lau et al., 2001, Science 294:858-862; Hutvagner et al., 2001, Science 293:834-838).MiRNA combines (Zeng et al. with the transcript sequence that only has the part complementarity, 2002, Molec.Cell 9:1327-1333), and under the situation that does not influence the steady state RNA level, prevent translation (Lee et al., 1993, Cell 75:843-854; Wightman et al., 1993, Cell 75:855-862).MiRNA and siRNA be by " cutting unit " processing, and with the composition of the reticent mixture of RNA inductive associate (Hutvagner et al., 2001, Science293:834-838; Grishok et al., 2001, Cell 106:23-34; Ketting et al., 2001, Genes Dev.15:2654-2659; Williams et al., 2002, Proc.Nntl.Acad.Sci.USA 99:6889-6894; Hammond et al., 2001, Science 293:1146-1150; Mourlatos et al., 2002, Genes Dev.16:720-728).A nearest report (Hutvagner et al., 2002, Sciencexpress 297:2056-2060) has been supposed only by determining by the adjusting of miRNA approach to the siRNA approach with the complementary degree of target transcript.Only infer that to have a siRNA of partial identity similar to miRNA to the mRNA target, will in translation repression, work, rather than cause RNA and degrade.
Also verified, siRNA and shRNA can be used for making in vivo gene silencing.Utilize siRNA and shRNA to carry out the ability of gene silencing in vivo, have the potential that makes it possible to select and develop siRNA for therepic use.Nearest report has been emphasized the potential treatment application of siRNA.The apoptosis of Fas mediation keeps liver with the hepatopathy spectrum is relevant widely and can pass through to suppress hepatocellular apoptosis death.(Song et al.2003, Nat.Medicine 9,347-351) due to the siRNA of Fas acceptor mouse carried out intravenous injection with target for Song.In mouse liver cell, at mRNA and protein level the Fas gene is carried out silence, stop apoptosis, and the protection mouse avoids hepatitis inductive liver destruction.Like this, reticent Fas expresses, and has kept avoiding the treatment prospect that cytotoxicity prevents liver injury by the protection liver cell.As another example, the siRNA that decides TNF-α with target carries out peritoneal injection to mouse.Suppress lipopolysaccharide-induced TNF-α genetic expression, and protected these mouse to avoid Sepsis.The concentrated area, these results suggest siRNA can work in vivo, and have potential as medicine (Sorensen et al., 2003, J.Mol.Biol.327,761-766).
People such as Martinez have reported and can disturb the selectivity target to decide oncogene mutation (Martinez et al., 2002, Proc.Natl.Acad.Sci.USA 99:14849-14854) with RNA.In this report, proved that the siRNA in zone of R248W mutant that target contains the p53 of point mutation surely makes the expression silencing of sudden change p53, but do not made the expression silencing of wild type p53.
Wilda etc. have reported that the siRNA that target can be decided the M-BCR/ABL fusion mRNA is used for removing leukemia cell's M-BCR/ABL mRNA and M-BRC/ABL cancer protein (Wilda et al., 2002, Oncogene 21:5716-5724).But this report also shows siRNA and Imatinib (a kind of small molecules ABL kinases tyrosine kinase inhibitors) united and is used for the leukemia cell, does not further increase apoptosis induced.
U.S. Patent No. 6,506,559 disclose the RNA interference method of the expression of target gene that is used for suppressing cell.This method comprise with have at double stranded region with target gene in the partially or completely double-stranded RNA transfered cell of the sequence that is equal to of sequence in or in the transfered cell external environment.RNA sequence with insertion, disappearance and simple point mutation with respect to target sequence also is found and can effectively suppresses to express.
It is the RNA interference of RNA fragment in the fruit bat vitro system of 21-23 Nucleotide (nt) that U.S. Patent Application Publication No.US2002/0086356 discloses employing length.This patent application has openly been instructed when with these 21-23nt fragment purifications and when being added back in the fruit bat extract, and the sequence-specific RNA when there is not long dsRNA in their mediations disturbs.This patent application has openly also been instructed, and also the oligonucleotide with chemosynthesis of same or similar character can be used for target and decide specific mrna, is used for degrading at mammalian cell.
It is that the double-stranded RNA (dsRNA) of 19-23nt is induced the sequence specific post transcriptional gene silencing in the fruit bat vitro system that PCT open WO02/44321 discloses length.This PCT has openly instructed by III type RNA enzyme sample processing reaction from the short interfering rna (siRNA) of long dsRNA generation or the siRNA duplex with 3 ' outstanding end of chemosynthesis, can mediate the effective target RNA cutting in the lysate, and cleavage site is positioned near the center in the guiding zone that siRNA crossed over.This PCT openly also provides evidence, proves that the direction of dsRNA processing has been determined to be cut with still antisense target RNA of justice by the siRNP mixture that produces.
U.S. Patent Application Publication No.US 2002/016216 discloses the method that weakens expression of target gene in the cultured cells, this be by with double-stranded RNA (dsRNA) realizing in the amount transfered cell that is enough to weaken expression of target gene, described double-stranded RNA is included under the stringent condition nucleotide sequence with the nucleotide sequence hybridization of target gene.
The open WO 03/006477 of PCT discloses the RNA precursor of through engineering approaches, when it is expressed in cell, process by cell, to produce the fixed siRNA (siRNA) of target, described siRNA disturbs (RNAi) approach optionally to make the fixed gene silencing of target (by cleavage specificity mRNA) with the RNA of cell self.This PCT has openly instructed in the nucleic acid molecule transfered cell by the RNA precursor of these through engineering approaches of will encoding with proper regulation sequence, can be on time and space, that is, in specified time and/or the optionally expression of the RNA precursor of control engineeringization in particular organization, organ or cell.
To the discussion of reference with quote, should not be interpreted as admitting that described reference is a prior art of the present invention herein.
3. summary of the invention
The invention provides and use RNA to disturb identified gene or its product and a kind of reagent, as the interaction between medicine and/or another kind of gene or its product, as causing death/the collaborative interactional method and composition that causes death.The present invention also provides collaborative the causing death of utilizing between STK6 kinases or TPX2 and the kinesin sample dynein KSP inhibitor to interact and treatment method for cancer and composition.The present invention also provides the gene that participates in DNA destructive cell response, and therepic use.
On the one hand, the invention provides the method for identified gene, the product of described gene is regulated the effect of a kind of reagent to a kind of cell of cell type.This method comprises that (a) makes a large amount of group of one or more cells of described cell type contact with described reagent, wherein the group of each described one or more cell comprises one or more the different siRNAs (siRNA) from a large amount of different siRNA, the gene that described one or more different siRNA target phasings are same, and described a large amount of different siRNA comprise the heterogeneic siRNA in the cell of distinguishing the fixed described cell type of target; (b) effect to the cell of the described cell type that do not comprise the fixed arbitrary described heterogeneic siRNA of target compares to the effect of the group of each described one or more cell and described reagent with described reagent; If (c) effect of the cell of the described cell type of arbitrary described heterogeneic siRNA is different calmly to not comprising target to the effect of the group of described one or more cells of described one or more different siRNA of comprising the fixed described gene of target and described reagent for described reagent, then identified gene is that described its product is regulated the gene of described reagent to the effect of the cell of described cell type.In one embodiment, comprise one or more each described groups of cells among described a large amount of siRNA by before described contact procedure, obtaining with described one or more siRNA transfections.In one embodiment, the component to each described one or more cell drives capable contact procedure (a) into.
In a kind of specific embodiment, the invention provides the method for identified gene, the product of described gene is regulated the effect of a kind of reagent to a kind of cell of cell type, described method comprises that (a) is with comprising from a large amount of group of one or more cells of the described cell type of composition transfection of one or more different siRNAs (siRNA) of a large amount of different siRNA each, the gene that described one or more different siRNA target phasings are same, and described a large amount of different siRNA comprise the heterogeneic siRNA in the cell of distinguishing the fixed described cell type of target; (b) described a large amount of group of one or more cells is contacted with described reagent; (c) effect to the cell of the described cell type of the fixed arbitrary described heterogeneic siRNA transfection of target of no use compares to the effect of the group of each described one or more cell and described reagent with described reagent; If (d) effect of the cell of the described cell type of arbitrary described heterogeneic siRNA is different calmly to not comprising target to the effect of the group of described one or more cells of described one or more different siRNA of comprising the fixed described gene of target and described reagent for described reagent, then identified gene is that described its product is regulated the gene of described reagent to the effect of the cell of described cell type.
With described reagent the effect of the cell of the described cell type that do not comprise the fixed arbitrary described heterogeneic siRNA of target is compared, described reagent can strengthen the effect of the group of each described one or more cell of comprising described one or more different siRNA.Perhaps, with described reagent the effect of the cell of the described cell type that do not comprise the fixed arbitrary described heterogeneic siRNA of target is compared, described reagent can weaken the effect of the group of each described one or more cell of comprising described one or more different siRNA.
Preferably, described reagent acts on by fixed arbitrary described different genes gene or its encoded protein in addition of described a large amount of siRNA targets.Preferably, described a large amount of siRNA comprise the fixed different siRNA of at least a described heterogeneic kind of k at least of target, and wherein said k is selected from 2,3,4,5,6 and 10.More preferably, described a large amount of siRNA comprise each the different siRNA of the kind of k at least at least two kinds of different genes in the fixed described different genes of target, and wherein said k is selected from 2,3,4,5,6 and 10.Still more preferably, described a large amount of siRNA comprise each the different siRNA of the kind of k at least in the fixed described different genes of target, and wherein said k is selected from 2,3,4,5,6 and 10.
Preferably, at least a, at least two kinds or each in described a large amount of different genes all comprises target phasing 2,3,4,5,6 or 10 kind of different siRNA with target gene.In a kind of preferred embodiment, total siRNA concentration of one or more siRNA approximately concentration with the single siRNA that uses separately is identical, for example 100nM.Preferably, total siRNA concentration of one or more siRNA is the optimal concentration that is used to make as the target gene silence of target.Optimal concentration is such concentration, when this concentration of further increase, does not increase reticent level substantially.In one embodiment, optimal concentration is such concentration, and when this concentration of further increase, the increase of reticent level is no more than 5%, 10% or 20%.In a kind of preferred embodiment, the ratio of the every kind of siRNA that comprises among one or more siRNA is identical.In another kind of preferred embodiment, the every kind of siRNA difference each other that comprises among one or more siRNA is less than 5%, 10%, 20% or 50%.In a kind of preferred embodiment, at least a among one or more siRNA accounts for more than 90%, 80%, 70%, 50% or 20% of total siRNA concentration of one or more siRNA.In another kind of preferred embodiment, there is not a kind of siRNA to account for more than 90%, 80%, 70%, 50% or 20% of total siRNA concentration of one or more siRNA among one or more siRNA.In a kind of preferred embodiment, select the composition of one or more siRNA, the concentration that comprises number He each siRNA of different siRNA, make one or more siRNA cause any not as the silence of the gene of target less than 30%, 20%, 10% or 5%, 1%, 0.1 or 0.01%.In another embodiment, the optimal concentration the when concentration of each siRNA among one or more siRNA all is lower than independent the use.In a kind of preferred embodiment, the concentration of at least a siRNA among one or more siRNA is lower than at least 30%, 50%, 75%, 80%, 85%, 90 or 95% siRNA concentration of the gene silencing that effectively reaches when not having other siRNA or not existing other to be designed for the siRNA that makes gene silencing.In another kind of preferred embodiment, the gene silencing that the concentration of each the different siRNA among one or more siRNA causes is lower than 30%, 20%, 10% or 5% of gene silencing when not having other siRNA or not existing other to be designed for the siRNA that makes gene silencing.In a kind of preferred embodiment, 30%, 20%, 10% or 5% of the target gene silence that the target gene silence that the concentration of every kind of siRNA causes causes when being lower than independent the use, and described a large amount of siRNA causes at least 80 or 90% of target gene silence.
In one embodiment, described cell type is the cancer cells type.In another embodiment, described effect is the growth-inhibiting effect.In a kind of specific embodiment, described reagent is the KSP inhibitor.In preferred embodiments, described different gene comprises at least 5 kinds, at least 10 kinds, at least 100 kinds or at least 1000 kinds of different genes.In one embodiment, described different gene is different native gene.
On the other hand, the invention provides the method that is used for identifying with a kind of interactional gene of elementary (primary) target gene of cell of cell type.This method comprises that (a) makes a large amount of group of one or more cells of described cell type contact with a kind of reagent, wherein said reagent is regulated described primary target expression of gene and/or by the proteic activity of described primary target genes encoding, and the group of wherein said cell comprises one or more the different siRNA among a large amount of different siRNA, described one or more different siRNA target phasings gene together, and described a large amount of different siRNA comprise the siRNA of different secondary (secondary) gene in the fixed described cell of difference target; (b) effect to the cell of the described cell type of the siRNA that do not comprise the fixed arbitrary described different secondary genes of target compares to the effect of the group of each described one or more cell and described reagent with described reagent; If (c) effect of the cell of the described cell type of the siRNA of arbitrary described different secondary genes is different calmly to not comprising target to the effect of the group of described one or more cells of one or more different siRNA of comprising the fixed described gene of target and described reagent for described reagent, then identified gene is the gene of the described primary target gene interaction in the cell of described and described cell type.In one embodiment, comprise one or more each described groups of cells among described a large amount of siRNA by before described contact procedure, obtaining with described one or more siRNA transfections.
In a kind of specific embodiment, the invention provides the method for the gene of the primary target gene interaction in the cell of identifying with a kind of cell type, described method comprises that (a) is with comprising from a large amount of group of one or more cells of the described cell type of composition transfection of one or more different siRNAs (siRNA) of a large amount of different siRNA each, the gene that described one or more different siRNA target phasings are same, and described a large amount of different siRNA comprise the heterogeneic siRNA in the cell of distinguishing the fixed described cell type of target; (b) described a large amount of group of one or more cells of described cell type is contacted with a kind of reagent, wherein said reagent is regulated described primary target expression of gene and/or by the proteic activity of described primary target genes encoding; (c) effect to the cell of the described cell type of the siRNA that do not comprise the fixed arbitrary described different secondary genes of target compares to the effect of the group of each described one or more cell and described reagent with described reagent; If (d) effect of the cell of the described cell type of the siRNA of arbitrary described different secondary genes is different calmly to not comprising target to the effect of the group of described one or more cells of one or more siRNA of comprising the fixed described gene of target and described reagent for described reagent, then identified gene is the gene of the described primary target gene interaction in the cell of described and described cell type.
In one embodiment, described reagent comprises the fixed described primary target gene of target and makes its reticent siRNA.In another embodiment, described reagent comprises 2,3,4,5,6 or 10 kind of different siRNA of the fixed described primary target gene of target.In a kind of preferred embodiment, each of described different siRNA is the fixed described primary target gene of target all.In a kind of preferred embodiment, total siRNA concentration of the described different siRNA approximately concentration with the single siRNA that uses separately is identical, for example 100nM.Preferably, total siRNA concentration of described different siRNA is the optimal concentration that is used to make the primary target gene silencing.Optimal concentration is such concentration, when this concentration of further increase, does not increase reticent level substantially.In one embodiment, optimal concentration is such concentration, and when this concentration of further increase, the increase of reticent level is no more than 5%, 10% or 20%.In a kind of preferred embodiment, the ratio of the every kind of siRNA that comprises among the different siRNA is identical.In another kind of preferred embodiment, the every kind of siRNA difference each other that comprises among the different siRNA is less than 5%, 10%, 20% or 50%.In a kind of preferred embodiment, at least a among the different siRNA accounts for more than 90%, 80%, 70%, 50% or 20% of total siRNA concentration of different siRNA.In another kind of preferred embodiment, there is not a kind of siRNA to account for more than 90%, 80%, 70%, 50% or 20% of total siRNA concentration of different siRNA among the different siRNA.In a kind of preferred embodiment, select the composition of different siRNA, the concentration that comprises number He each siRNA of different siRNA, make different siRNA cause any not as the silence of the gene of target less than 30%, 20%, 10% or 5%, 1%, 0.1 or 0.01%.In another embodiment, the optimal concentration the when concentration of each siRNA all is lower than independent the use.In a kind of preferred embodiment, the concentration of at least a siRNA is lower than at least 30%, 50%, 75%, 80%, 85%, 90 or 95% siRNA concentration of the gene silencing that effectively reaches when not having other siRNA or not existing other to be designed for the siRNA that makes gene silencing.In another kind of preferred embodiment, the gene silencing that the concentration of each different siRNA causes is lower than 30%, 20%, 10% or 5% of gene silencing when not having other siRNA or not existing other to be designed for the siRNA that makes gene silencing.In a kind of preferred embodiment, 30%, 20%, 10% or 5% of the target gene silence that the target gene silence that the concentration of every kind of siRNA causes causes when being lower than independent the use, and all siRNA cause at least 80 or 90% of target gene silence together.In another embodiment, described reagent comprises the proteic inhibitor by described primary target genes encoding.
With described reagent the effect of the cell of the described cell type of the siRNA that do not comprise the fixed arbitrary described different secondary genes of target is compared, described reagent can strengthen the effect of the group of described one or more cells.Perhaps, with described reagent the effect of the cell of the described cell type of the siRNA that do not comprise the fixed arbitrary described different secondary genes of target is compared, described reagent can weaken the effect of the group of described one or more cells.
Preferably, one or more siRNA at least a, at least two kinds or each in described a large amount of different genes comprise target phasing 2,3,4,5,6 or 10 kind of different siRNA with target gene.In a kind of preferred embodiment, the target phasing is identical with the about concentration with the single siRNA that uses separately of total siRNA concentration of one or more siRNA of target gene, for example 100nM.Preferably, total siRNA concentration of one or more siRNA is the optimal concentration that is used to make as the target gene silence of target.Optimal concentration is such concentration, when this concentration of further increase, does not increase reticent level substantially.In one embodiment, optimal concentration is such concentration, and when this concentration of further increase, the increase of reticent level is no more than 5%, 10% or 20%.In a kind of preferred embodiment, the ratio of the every kind of siRNA that comprises among one or more siRNA is identical.In another kind of preferred embodiment, the every kind of siRNA difference each other that comprises among one or more siRNA is less than 5%, 10%, 20% or 50%.In a kind of preferred embodiment, at least a among one or more siRNA accounts for more than 90%, 80%, 70%, 50% or 20% of total siRNA concentration of one or more siRNA.In another kind of preferred embodiment, there is not a kind of siRNA to account for more than 90%, 80%, 70%, 50% or 20% of total siRNA concentration of one or more siRNA among one or more siRNA.In a kind of preferred embodiment, select the composition of one or more siRNA, the concentration that comprises number He each siRNA of different siRNA, make one or more siRNA cause any not as the silence of the gene of target less than 30%, 20%, 10% or 5%, 1%, 0.1 or 0.01%.In another embodiment, the optimal concentration the when concentration of each siRNA among one or more siRNA all is lower than independent the use.In a kind of preferred embodiment, the concentration of at least a siRNA among one or more siRNA is lower than at least 30%, 50%, 75%, 80%, 85%, 90 or 95% siRNA concentration of the gene silencing that effectively reaches when not having other siRNA or not existing other to be designed for the siRNA that makes gene silencing.In another kind of preferred embodiment, the gene silencing that the concentration of each the different siRNA among one or more siRNA causes is lower than 30%, 20%, 10% or 5% of gene silencing when not having other siRNA or not existing other to be designed for the siRNA that makes gene silencing.In a kind of preferred embodiment, 30%, 20%, 10% or 5% of the target gene silence that the target gene silence that the concentration of every kind of siRNA causes causes when being lower than independent the use, and described a large amount of siRNA causes at least 80 or 90% of target gene silence.
In one embodiment, the group of each described one or more cell is by obtaining with described one or more different siRNA transfections before described contact procedure.In another embodiment, described primary target is KSP.In preferred embodiments, described different secondary gene comprises at least 5 kinds, at least 10 kinds, at least 100 kinds or at least 1000 kinds of different genes.In one embodiment, described different secondary gene is different native gene.In one embodiment, described cell type is the cancer cells type.
On the other hand, the invention provides and be used for the treatment of the mammiferous method of suffering from cancer, comprise a kind of reagent to described administration treatment q.s, described reagent is regulated STK6 or TPX2 expression of gene and/or by the proteic activity of described STK6 or TPX2 genes encoding, wherein said Mammals carries out a kind of treatment, and described treatment comprises the KSP inhibitor to described administration treatment q.s.The present invention also provides and has been used for the treatment of the mammiferous method of suffering from cancer, comprise to described administration i) treatment q.s a kind of reagent, described reagent is regulated STK6 or TPX2 expression of gene and/or by the proteic activity of described STK6 or TPX2 genes encoding with ii) treat the KSP inhibitor of q.s.In one embodiment, described reagent reduces STK6 or TPX2 expression of gene described in the cell of described cancer.In a kind of preferred embodiment, described reagent comprises the siRNA of fixed described STK6 of target or TPX2 gene.In another embodiment, described Mammals is the people, and described siRNA can be selected from the siRNA shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and the SEQ ID NO:6.In another kind of preferred embodiment, described reagent comprises the siRNA of the fixed described TPX2 gene of target.In another embodiment, described Mammals is the people, and described siRNA can be selected from the siRNA shown in SEQ ID NO:1237, SEQ ID NO:1238 and the SEQID NO:1239.
In another embodiment, the invention provides and be used for the treatment of the mammiferous method of suffering from cancer, comprise to described administration i) treatment q.s first kind of reagent, described first kind of reagent is regulated STK6 or TPX2 expression of gene and/or by the proteic activity of described STK6 or TPX2 genes encoding, ii) treat second kind of reagent of q.s, described second kind of reagent is regulated the KSP expression of gene and/or by the proteic activity of described KSP genes encoding.In a kind of preferred embodiment, described first kind of reagent comprises the siRNA of fixed described TPX2 of target or TPX2 gene, and described second kind of reagent comprises the siRNA of the fixed described KSP gene of target.In another kind of preferred embodiment, described Mammals is the people, and wherein said siRNA is selected from the siRNA shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and the SEQ ID NO:6.In another kind of preferred embodiment, described reagent comprises the siRNA of the fixed described TPX2 gene of target.In another embodiment, described Mammals is the people, and described siRNA can be selected from the siRNA shown in SEQ ID NO:1237, SEQ ID NO:1238 and the SEQ ID NO:1239.
In another embodiment, the invention provides and be used to assess the method for cell the Growth Inhibition resistance of KSP inhibitor, described method comprises STK6 or the TPX2 expression of gene level of determining in the described cell, the described expression level that wherein is higher than predetermined threshold levels shows that described cell has resistance to the growth-inhibiting effect of described KSP inhibitor.In a kind of preferred embodiment, by comprising the method for measuring described STK6 or TPX2 expression of gene level with one or more nucleotide probes, determine described STK6 or TPX2 expression of gene level, each in described one or more polynucleotide probes all comprises the nucleotide sequence in described STK6 or the TPX2 gene.Described one or more polynucleotide probes can be the polynucleotide probes on microarray.
In another embodiment, the invention provides and be used to assess the method for cell the Growth Inhibition resistance of KSP inhibitor, described method comprises the proteic abundance level of determining in the described cell by STK6 or TPX2 genes encoding, the described proteic described abundance level that wherein is higher than predetermined threshold levels shows that described cell has resistance to the growth-inhibiting effect of described KSP inhibitor.The present invention also provides and has been used to assess the method for cell to the Growth Inhibition resistance of KSP inhibitor, described method comprises the proteic activity level of determining in the described cell by STK6 or TPX2 genes encoding, the described activity level that wherein is higher than predetermined threshold levels shows that described cell has resistance to the growth-inhibiting effect of described KSP inhibitor.In a kind of preferred embodiment, described cell is people's cell.
In another embodiment, the invention provides and be used to regulate the method for cell the Growth Inhibition resistance of KSP inhibitor, comprise a kind of reagent that makes described cells contacting q.s, described reagent is regulated STK6 or TPX2 expression of gene and/or by the proteic activity of described STK6 or TPX2 genes encoding.The present invention also provides and has been used for regulating the method for Mammals cell to the Growth Inhibition resistance of KSP inhibitor, comprise a kind of reagent to described administration treatment q.s, described reagent is regulated STK6 or TPX2 expression of gene and/or by the proteic activity of described STK6 or TPX2 genes encoding.The present invention also provides the method that is used to regulate the cell growth, comprise and make described cells contacting i) a kind of reagent of q.s, described reagent is regulated STK6 or TPX2 expression of gene and/or by the proteic activity of described STK6 or TPX2 genes encoding and the ii) KSP inhibitor of q.s.Preferably, described reagent reduces STK6 described in the described cell or TPX2 expression of gene.In a kind of preferred embodiment, described reagent comprises the siRNA of the fixed described STK6 gene of target.In another embodiment, described cell is people's cell, and wherein said siRNA is selected from the siRNA shown in SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5 and the SEQ ID NO:6.In another kind of preferred embodiment, described reagent comprises the siRNA of the fixed described TPX2 gene of target.In another embodiment, described cell is people's cell, and described siRNA can be selected from the siRNA shown in SEQ ID NO:1237, SEQID NO:1238 and the SEQ ID NO:1239.
In another embodiment, the invention provides and be used to identify and regulate the compositions and methods of cell the Growth Inhibition resistance of KSP inhibitor, wherein said reagent can be regulated STK6 or TPX2 expression of gene and/or by the proteic activity of described STK6 or TPX2 genes encoding, described method comprise described KSP inhibitor when relatively having described reagent to the cell inhibiting effect of expressing described STK6 or TPX2 gene when not having described reagent described KSP inhibitor to expressing the cell inhibiting effect of described STK6 or TPX2 gene, the described inhibiting difference of wherein said KSP inhibitor identifies that described reagent can regulate the Growth Inhibition resistance of described cell to the KSP inhibitor.
The present invention also provides and has been used to identify and can regulates the compositions and methods of cell to the Growth Inhibition resistance of KSP inhibitor, wherein said reagent can be regulated STK6 or TPX2 expression of gene and/or by the proteic activity of described STK6 or TPX2 genes encoding, described method comprises: (a) first kind of cell of expressing described STK6 or TPX2 gene contacted existing with described KSP inhibitor, and measure first kind of growth-inhibiting effect; (b) second kind of cell of expressing described STK6 or TPX2 gene contacted with described KSP inhibitor, and measure second kind of growth-inhibiting effect; (c) compare in described step (a) and described first kind and second kind of restraining effect of measuring (b), difference between wherein said first kind and the second kind of restraining effect identifies that described reagent can regulate the Growth Inhibition resistance of cell to the KSP inhibitor.In a kind of preferred embodiment, described reagent is the molecule that reduces described STK6 or TPX2 genetic expression.In another kind of preferred embodiment, described reagent comprises the siRNA of the fixed described STK6 gene of target.In another kind of preferred embodiment, described cell is people's cell, and wherein said siRNA is selected from the siRNA shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5 and the SEQ ID NO:6.In another kind of preferred embodiment, described reagent comprises the siRNA of the fixed described TPX2 gene of target.In another embodiment, described cell is people's cell, and described siRNA can be selected from the siRNA shown in SEQID NO:1237, SEQ ID NO:1238 and the SEQ ID NO:1239.
On the other hand, the invention provides the cell that comprises one or more different siRNAs (siRNA), STK6 or TPX2 gene in the fixed described cell of described siRNA target.Described cell can be people's cell.Described cell also can be the mouse cell.In one embodiment, described cell is people's cell, and among described one or more different siRNA each all is selected from the siRNA shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5 and the SEQ ID NO:6.In another embodiment, described cell is people's cell, and described siRNA can be selected from the siRNA shown in SEQ IDNO:1237, SEQ ID NO:1238 and the SEQ ID NO:1239.In one embodiment, described cell produces by the composition transfection of using described one or more different siRNA, total siRNA concentration of wherein said composition is the optimal concentration that is used to make described STK6 or TPX2 gene silencing, wherein said optimal concentration is such concentration, when this concentration of further increase, do not increase reticent level substantially.In one embodiment, described optimal concentration is such concentration, and when this concentration of further increase, the increase of reticent level is no more than more than 20%, more than 10% or 5%.In one embodiment, the concentration of every kind of described different siRNA is approximately identical.In one embodiment, every kind of described different siRNA concentration separately difference each other is less than 50%, less than 20% or less than 10%.In another embodiment, do not have in the described composition a kind of siRNA account for more than 80% of described total siRNA concentration of described different siRNA, more than 50% or more than 20%.In another embodiment, the concentration of at least a siRNA in the described composition is more than 20% or more than 50% of described total siRNA concentration of described different siRNA.In another embodiment, select the concentration of number He each siRNA of different siRNA in the described composition, make described composition cause any not as the silence of the gene of target less than 10%, less than 1%, less than 0.1% or less than 0.01%.
On the other hand, the invention provides and be used for the microarray of diagnosis cell the Growth Inhibition resistance of KSP inhibitor.Described microarray comprises one or more polynucleotide probes, and wherein each described polynucleotide probes comprises the nucleotide sequence in STK6 or the TPX2 gene.
On the other hand, the invention provides and be used for the test kit of diagnosis cell the Growth Inhibition resistance of KSP inhibitor.Described test kit comprises one or more polynucleotide probes that place one or more containers, and wherein each described polynucleotide probes comprises the nucleotide sequence in STK6 or the TPX2 gene.The present invention also provides the test kit that is used to screen a kind of reagent, and described reagent is regulated the Growth Inhibition resistance of cell to the KSP inhibitor.Described test kit comprises the cell that (i) that place one or more containers comprises one or more different siRNAs (siRNA), STK6 or TPX2 gene in the fixed described cell of described siRNA target; (ii) KSP inhibitor.On the other hand, the invention provides and be used for the treatment of the mammiferous test kit of suffering from cancer, this test kit comprises the adjusting STK6 of (i) q.s that places one or more containers or TPX2 expression of gene and/or by the proteic active reagent of described STK6 or TPX2 genes encoding; (ii) KSP inhibitor.
In the present invention, the KSP inhibitor can be (1S)-1-{[(2S)-4-(2, the 5-difluorophenyl)-2-phenyl-2 of describing among the PCT application PCT/US03/18482 that submitted on June 12nd, 2003,5-dihydro-1H-pyrroles-1-yl] carbonyl }-2-methyl propylamine.
The present invention also provides the method that is used for identifying with a kind of gene of primary target gene interaction of cell of cell type.This method comprises that (a) makes a kind of reagent of one or more cells contacting of described cell type, wherein said reagent is regulated the secondary target expression of gene and/or by the proteic activity of described secondary target genes encoding, and first siRNA (siRNA) of the fixed described primary target gene of wherein said one or more cell expressing targets; (b) more described reagent is to the effect to the cell of the described cell type of not expressing a described siRNA of the effect of described one or more cells of described clone and described reagent; If (c) described reagent is different to the effect of the cell of the described cell type of not expressing a described siRNA to the effect of described one or more cells of expressing a described siRNA and described reagent, then identify described secondary target gene for the cell of described cell type in the gene of primary target gene interaction.
In a kind of particular, described method comprises that (a) produces the expression target clone of the cell of the described cell type of first siRNA (siRNA) of described primary target gene calmly; (b) make a kind of reagent of one or more cells contacting of described clone, wherein said reagent is regulated the secondary target expression of gene and/or by the proteic activity of described secondary target genes encoding; (c) more described reagent is to the effect to the cell of the described cell type of not expressing a described siRNA of the effect of described one or more cells of described clone and described reagent; If (d) described reagent is different to the effect of the cell of the described cell type of not expressing a described siRNA to the effect of described one or more cells of expressing a described siRNA and described reagent, then identify described secondary target gene for the cell of described cell type in the gene of primary target gene interaction.
In some embodiments, with described reagent the effect of the cell of the described cell type of not expressing a described siRNA is compared, described reagent strengthens the effect of described one or more cells of expressing a described siRNA.In some embodiments, with described reagent the effect of the cell of the described cell type of not expressing a described siRNA is compared, described reagent weakens the effect of described one or more cells of expressing a described siRNA.In one embodiment, described reagent is the inhibitor of described secondary target gene.The effect of described reagent can be the change of the cell of described cell type to the susceptibility of medicine or ionizing rays, described medicine such as DNA disrupting agent are as the topoisomerase I inhibitor, as camptothecine, topoisomerase II inhibitor, as Zorubicin, DNA wedding agent, as cis-platinum and antimetabolite.
In another embodiment, described reagent comprises the fixed described secondary target gene of one or more targets and makes the 2nd siRNA of its silence.Preferably, described one or more comprise the different siRNA of k kind at least, as at least 2,3,4,5,6 or 10 kind of different siRNA.In a kind of preferred embodiment, total siRNA concentration of one or more the 2nd siRNA approximately concentration with the single siRNA that uses separately is identical, for example 100nM.Preferably, total siRNA concentration of one or more the 2nd siRNA is the optimal concentration that is used to make as the secondary target gene silencing of target.Optimal concentration is such concentration, when this concentration of further increase, does not increase reticent level substantially.In one embodiment, optimal concentration is such concentration, and when this concentration of further increase, the increase of reticent level is no more than 5%, 10% or 20%.In a kind of preferred embodiment, the ratio of the every kind of siRNA that comprises among one or more the 2nd siRNA is identical.In another kind of preferred embodiment, the every kind of siRNA difference each other that comprises among one or more the 2nd siRNA is less than 5%, 10%, 20% or 50%.In a kind of preferred embodiment, at least a among one or more the 2nd siRNA accounts for more than 90%, 80%, 70%, 50% or 20% of total siRNA concentration of one or more siRNA.In another kind of preferred embodiment, there is not a kind of siRNA to account for more than 90%, 80%, 70%, 50% or 20% of total siRNA concentration of one or more the 2nd siRNA among one or more siRNA.In a kind of preferred embodiment, select the composition of one or more siRNA, the concentration that comprises number He each the 2nd siRNA of different siRNA, make one or more the 2nd siRNA cause any not as the silence of the gene of target less than 30%, 20%, 10% or 5%, 1%, 0.1 or 0.01%.In another embodiment, the optimal concentration the when concentration of each siRNA among one or more the 2nd siRNA all is lower than independent the use.In a kind of preferred embodiment, the concentration of at least a siRNA among one or more siRNA is lower than at least 30%, 50%, 75%, 80%, 85%, 90 or 95% siRNA concentration of the gene silencing that effectively reaches when not having other siRNA or not existing other to be designed for the siRNA that makes gene silencing.In another kind of preferred embodiment, the gene silencing that the concentration of each the different siRNA among one or more the 2nd siRNA causes is lower than 30%, 20%, 10% or 5% of gene silencing when not having other siRNA or not existing other to be designed for the siRNA that makes gene silencing.In a kind of preferred embodiment, 30%, 20%, 10% or 5% of the secondary target gene silencing that the target gene silence that the concentration of every kind of siRNA causes causes when being lower than independent the use, and described a large amount of siRNA causes at least 80 or 90% of secondary target gene silencing.
In one embodiment, described cell is the cancer cells type.In another embodiment, described primary target gene is p53.
In a kind of preferred embodiment, in a large amount of different secondary target genes each, the step of repetition methods (b)-(d).A large amount of secondary target genes can comprise at least 5,10,100,1000 with 5000 kinds of different genes.
The present invention also provides and has been used for the treatment of the mammiferous method of suffering from cancer.This method comprises a kind of reagent to described administration treatment q.s, described reagent is regulated a kind of expression of gene and/or by the proteic activity of described genes encoding, wherein said Mammals carries out a kind of treatment, and described treatment comprises the composition that comprises one or more DNA disrupting agents to described administration treatment q.s.In one embodiment, the invention provides and be used for the treatment of the mammiferous method of suffering from cancer, comprise to described administration i) treatment q.s a kind of reagent, described reagent is regulated a kind of expression of gene and/or by the proteic activity of described genes encoding with ii) treat the composition that comprises one or more DNA disrupting agents of q.s.
Preferably, described reagent makes the expression decreased of described gene in the cell of described cancer.In a kind of preferred embodiment, described reagent comprises the siRNA of the fixed described gene of target.In specific embodiments, described gene is EPHB3, WEE1, ELK1, STK6, CHEK1 or BRCA2.Described reagent also can be to strengthen the reagent that described gene is expressed in the cell of described cancer.Described one or more DNA disrupting agents can comprise the topoisomerase I inhibitor, as camptothecine, topoisomerase II inhibitor, as Zorubicin, DNA wedding agent, as cis-platinum, antimetabolite or ionizing rays.
The present invention also provides and has been used to assess the method for cell to the Growth Inhibition susceptibility of DNA disrupting agent.Described method comprises a kind of gene transcription level of determining in the described cell, wherein is lower than the described transcriptional level of predetermined threshold levels, shows that described cell has susceptibility to the growth-inhibiting effect of described DNA disrupting agent.Described DNA disrupting agent can be the topoisomerase I inhibitor, as camptothecine, topoisomerase II inhibitor, as Zorubicin, DNA wedding agent, as cis-platinum, antimetabolite or ionizing rays.In a kind of preferred embodiment, described gene is EPHB3, WEE1, ELK1, STK6, CHEK1 or BRCA2.In a kind of preferred embodiment, by comprising the method for measuring described gene transcription level with one or more nucleotide probes, determine described gene transcription level, each in described one or more polynucleotide probes all comprises the nucleotide sequence in the described gene.In one embodiment, described one or more polynucleotide probes are the polynucleotide probes on microarray.
In another embodiment, the invention provides and be used to assess cell, as the method for people's cell to the Growth Inhibition susceptibility of DNA disrupting agent.Described method comprises to be determined in the described cell wherein to be lower than the described proteic described abundance level of predetermined threshold levels by a kind of proteic abundance level of genes encoding, shows that described cell has susceptibility to the growth-inhibiting effect of described DNA disrupting agent.The present invention also provides and has been used to assess cell, as the method for people's cell to the Growth Inhibition susceptibility of DNA disrupting agent, described method comprises the proteic activity level of determining in the described cell by described genes encoding, the described activity level that wherein is higher than predetermined threshold levels shows that described cell has susceptibility to the growth-inhibiting effect of described DNA disrupting agent.The DNA disrupting agent can be the topoisomerase I inhibitor, as camptothecine, topoisomerase II inhibitor, as Zorubicin, DNA wedding agent, as cis-platinum, antimetabolite or ionizing rays.In a kind of preferred embodiment, described gene is EPHB3, WEE1, ELK1, STK6, CHEK1 or BRCA2.
The present invention also provides and has been used to regulate the method for cell to the susceptibility of DNA disrupting agent.This method comprises makes described cell contact with a kind of reagent of q.s, and described reagent is regulated the expression of gene that is selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51 and/or by the proteic activity of described genes encoding.The present invention also provides the method that is used to regulate the cell growth, comprise and make described cells contacting i) a kind of reagent of q.s, described reagent is regulated the expression of gene that is selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51 and/or by the proteic activity of described genes encoding; The ii) DNA disrupting agent of q.s.The DNA disrupting agent can be the topoisomerase I inhibitor, as camptothecine, topoisomerase II inhibitor, as Zorubicin, DNA wedding agent, as cis-platinum, antimetabolite or ionizing rays.
In one embodiment, described reagent reduces expression of gene described in the described cell.In a kind of preferred embodiment, described reagent comprises the siRNA of the fixed described gene of target.In another kind of preferred embodiment, described reagent comprises 2,3,4,5,6 or 10 kind of different siRNA of the fixed described gene of target.In a kind of preferred embodiment, total siRNA concentration of the different siRNA of the fixed described gene of the target approximately concentration with the single siRNA that uses separately is identical, for example 100nM.Preferably, the total concn of the different siRNA of the fixed described gene of target is the optimal concentration that is used to make described gene silencing.Optimal concentration is such concentration, when this concentration of further increase, does not increase reticent level substantially.In one embodiment, optimal concentration is such concentration, and when this concentration of further increase, the increase of reticent level is no more than 5%, 10% or 20%.In a kind of preferred embodiment, the ratio of the every kind of siRNA that comprises among the different siRNA is identical.In another kind of preferred embodiment, the every kind of siRNA difference each other that comprises among the different siRNA is less than 5%, 10%, 20% or 50%.In a kind of preferred embodiment, at least a among the different siRNA accounts for more than 90%, 80%, 70%, 50% or 20% of total siRNA concentration of different siRNA.In another kind of preferred embodiment, there is not a kind of siRNA to account for more than 90%, 80%, 70%, 50% or 20% of total siRNA concentration of different siRNA among the different siRNA.In a kind of preferred embodiment, select the composition of different siRNA, the concentration that comprises number He each siRNA of different siRNA, make different siRNA cause any not as the silence of the gene of target less than 30%, 20%, 10% or 5%, 1%, 0.1 or 0.01%.Optimal concentration when in other embodiments, the concentration of each siRNA among the different siRNA all is lower than independent the use.In a kind of preferred embodiment, the concentration of at least a siRNA among the different siRNA is lower than at least 30%, 50%, 75%, 80%, 85%, 90 or 95% siRNA concentration of the gene silencing that effectively reaches when not having other siRNA or not existing other to be designed for the siRNA that makes gene silencing.In another kind of preferred embodiment, the gene silencing that the concentration of each different siRNA causes is lower than 30%, 20%, 10% or 5% of gene silencing when not having other siRNA or not existing other to be designed for the siRNA that makes gene silencing.In a kind of preferred embodiment, 30%, 20%, 10% or 5% of the target gene silence that the gene silencing that the concentration of every kind of siRNA causes causes when being lower than independent the use, and a large amount of siRNA causes at least 80 or 90% of target gene silence.
The present invention also provides and has been used to identify and can regulates the compositions and methods of cell to the Growth Inhibition susceptibility of DNA disrupting agent, wherein said reagent can be regulated and be selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, the expression of gene of BARD1 and RAD51 and/or by the proteic activity of described genes encoding, this method comprise described DNA disrupting agent when relatively having described reagent to the cell inhibiting effect of expressing said gene when not having described reagent described DNA disrupting agent to the cell inhibiting effect of expressing said gene, the described inhibiting difference of wherein said DNA disrupting agent identifies that described reagent can regulate the Growth Inhibition susceptibility of described cell to the DNA disrupting agent.In one embodiment, the invention provides and be used to identify and regulate the compositions and methods of cell the Growth Inhibition susceptibility of DNA disrupting agent, wherein said reagent can be regulated the expression of gene that is selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51 and/or by the proteic activity of described genes encoding, this method comprises that (a) exists the cell that makes first kind of expressing said gene under the condition of described reagent to contact with described DNA disrupting agent, and measures first kind of growth-inhibiting effect; (b) cell of second kind of expressing said gene is contacted with described DNA disrupting agent, and measure second kind of growth-inhibiting effect; (c) compare in described step (a) and described first kind and second kind of restraining effect of measuring (b), difference between wherein said first kind and the second kind of restraining effect identifies that described reagent can regulate the Growth Inhibition susceptibility of described cell to the DNA disrupting agent.
Preferably, described cell expressing target is decided the siRNA of primary target gene.In one embodiment, described primary target gene is p53.
In a kind of preferred embodiment, described reagent is the molecule that reduces described genetic expression.In a kind of preferred embodiment, described reagent comprises the siRNA of the fixed described gene of target.In another kind of preferred embodiment, described reagent comprises 2,3,4,5,6 or 10 kind of different siRNA of the fixed described gene of target.In a kind of preferred embodiment, total siRNA concentration of the different siRNA of the fixed described gene of the target approximately concentration with the single siRNA that uses separately is identical, for example 100nM.Preferably, the total concn of the different siRNA of the fixed described gene of target is the optimal concentration that is used to make described gene silencing.Optimal concentration is such concentration, when this concentration of further increase, does not increase reticent level substantially.In one embodiment, optimal concentration is such concentration, and when this concentration of further increase, the increase of reticent level is no more than 5%, 10% or 20%.In a kind of preferred embodiment, the ratio of the every kind of siRNA that comprises among the different siRNA is identical.In another kind of preferred embodiment, the every kind of siRNA difference each other that comprises among the different siRNA is less than 5%, 10%, 20% or 50%.In a kind of preferred embodiment, at least a among the different siRNA accounts for more than 90%, 80%, 70%, 50% or 20% of total siRNA concentration of different siRNA.In another kind of preferred embodiment, there is not a kind of siRNA to account for more than 90%, 80%, 70%, 50% or 20% of total siRNA concentration of different siRNA among the different siRNA.In a kind of preferred embodiment, select the composition of different siRNA, the concentration that comprises number He each siRNA of different siRNA, make different siRNA cause any not as the silence of the gene of target less than 30%, 20%, 10% or 5%, 1%, 0.1 or 0.01%.In other scheme, the optimal concentration the when concentration of each siRNA among the different siRNA all is lower than independent the use.In a kind of preferred embodiment, the concentration of at least a siRNA among the different siRNA is lower than at least 30%, 50%, 75%, 80%, 85%, 90 or 95% siRNA concentration of the gene silencing that effectively reaches when not having other siRNA or not existing other to be designed for the siRNA that makes gene silencing.In another kind of preferred embodiment, the gene silencing that the concentration of each different siRNA causes is lower than 30%, 20%, 10% or 5% of gene silencing when not having other siRNA or not existing other to be designed for the siRNA that makes gene silencing.In a kind of preferred embodiment, 30%, 20%, 10% or 5% of the target gene silence that the gene silencing that the concentration of every kind of siRNA causes causes when being lower than independent the use, and a large amount of siRNA causes at least 80 or 90% of target gene silence.
In described method, described DNA disrupting agent can be the topoisomerase I inhibitor, as camptothecine, topoisomerase II inhibitor, as Zorubicin, DNA wedding agent, as cis-platinum, antimetabolite or ionizing rays.
The present invention also provides the cell that comprises one or more different siRNAs (siRNA), the target gene that is selected from EPHB3, WEE1, ELK1, BRCA1, BRCA2, BARD1 and RAD51 in the fixed described cell of described siRNA target.In one embodiment, described one or more siRNA comprise 2,3,4,5,6 or 10 kind of different siRNA.In a kind of preferred embodiment, total siRNA concentration of described one or more siRNA approximately concentration with the single siRNA that uses separately is identical, for example 100nM.Preferably, the total concn of described one or more siRNA is the optimal concentration that is used to make as the target gene silence of target.Optimal concentration is such concentration, when this concentration of further increase, does not increase reticent level substantially.In one embodiment, optimal concentration is such concentration, and when this concentration of further increase, the increase of reticent level is no more than 5%, 10% or 20%.In a kind of preferred embodiment, the ratio of the every kind of siRNA that comprises among described one or more siRNA is identical.In another kind of preferred embodiment, the every kind of siRNA difference each other that comprises among described one or more siRNA is less than 5%, 10%, 20% or 50%.In a kind of preferred embodiment, at least a among described one or more siRNA accounts for more than 90%, 80%, 70%, 50% or 20% of total siRNA concentration of described one or more siRNA.In another kind of preferred embodiment, there is not a kind of siRNA to account for more than 90%, 80%, 70%, 50% or 20% of total siRNA concentration of described one or more siRNA among described one or more siRNA.In a kind of preferred embodiment, select the composition of described one or more siRNA, the concentration that comprises number He each siRNA of different siRNA, make described one or more siRNA cause any not as the silence of the gene of target less than 30%, 20%, 10% or 5%, 1%, 0.1 or 0.01%.In other scheme, the optimal concentration the when concentration of each siRNA among described one or more siRNA all is lower than independent the use.In a kind of preferred embodiment, the concentration of at least a siRNA among described one or more siRNA is lower than at least 30%, 50%, 75%, 80%, 85%, 90 or 95% siRNA concentration of the gene silencing that effectively reaches when not having other siRNA or not existing other to be designed for the siRNA that makes gene silencing.In another kind of preferred embodiment, the gene silencing that the concentration of each the different siRNA among described one or more siRNA causes is lower than 30%, 20%, 10% or 5% of gene silencing when not having other siRNA or not existing other to be designed for the siRNA that makes gene silencing.In a kind of preferred embodiment, 30%, 20%, 10% or 5% of the target gene silence that the target gene silence that the concentration of every kind of siRNA causes causes when being lower than independent the use, and a large amount of siRNA causes at least 80 or 90% of target gene silence.
The present invention also provides and has been used for the microarray of diagnosis cell to the Growth Inhibition susceptibility of DNA disrupting agent.Described microarray comprises one or more polynucleotide probes, and wherein each described polynucleotide probes comprises the nucleotide sequence in one or more genes that are selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51.
The present invention also provides and has been used for the test kit of diagnosis cell to the Growth Inhibition susceptibility of DNA disrupting agent.Described test kit comprises one or more polynucleotide probes that place one or more containers, and wherein each described polynucleotide probes comprises the nucleotide sequence of the gene that is selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51.
The present invention also provides and has been used to screen the test kit of adjusting cell to the reagent of the Growth Inhibition susceptibility of DNA disrupting agent.Described test kit comprises the cell that (i) that place one or more containers comprises one or more different siRNAs (siRNA), the gene that is selected from EPHB3, WEE1, ELK1, BRCA1, BRCA2, BARD1 and RAD51 in the fixed described cell of described siRNA target; (ii) described DNA disrupting agent.
The present invention also provides and has been used for the treatment of the mammiferous test kit of suffering from cancer, described test kit comprises a kind of reagent of (i) q.s that places one or more containers, and described reagent is regulated the expression of gene that is selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51 and/or by the proteic activity of described genes encoding; (ii) described DNA disrupting agent.
In test kit of the present invention, described DNA disrupting agent can be the topoisomerase I inhibitor, as camptothecine, topoisomerase II inhibitor, as Zorubicin, DNA wedding agent, as cis-platinum, antimetabolite or ionizing rays.
The present invention also provides and has been used to assess the reactive method of a kind of cell of cell type to pharmacological agent, comprise that (a) makes the described medicine of one or more cells contacting of described cell type, wherein said one or more cell expressing targets are decided first siRNA (siRNA) of primary target gene, and wherein said one or more cells are accepted a kind of processing of composition, and described composition is regulated one or more secondary target expression of gene and/or respectively by the proteic activity of described one or more secondary target genes encodings; (b) make the described medicine of one or more cells contacting of described cell type, wherein said one or more cells are not expressed the siRNA (siRNA) of the fixed described primary target gene of target, and wherein said one or more cells are accepted the governing stage target gene expression and/or by the processing of the proteic active described reagent of described secondary target genes encoding; (c) the described medicine of relatively measuring in step (a) is to the effect to described one or more cells of the described medicine of the effect of described one or more cells and measurement in step (b), thereby assesses the reactivity of described cell to described pharmacological agent.In one embodiment, described method further comprises the step (d) to each repeating step (a)-(b) in the different in a large number secondary target genes.
In a kind of particular, the invention provides and be used to assess the reactive method of a kind of cell of cell type to pharmacological agent, this method comprises that (a) preparation expresses the clone of cell of described subclass type that target is decided first siRNA (siRNA) of primary target gene; (b) make the described clone's who expresses a described siRNA the described medicine of one or more cells contacting, wherein said one or more cells are accepted the governing stage target gene expression and/or by the processing of the proteic active reagent of described secondary target genes encoding; (c) make the described medicine of one or more cells contacting of the described cell type of the siRNA (siRNA) of not expressing the fixed described primary target gene of target, wherein said one or more cells are accepted the governing stage target gene expression and/or by the processing of the proteic active described reagent of described secondary target genes encoding; (d) the described medicine of relatively measuring in step (b) is to the effect to described one or more cells of the described medicine of the effect of described one or more cells and measurement in step (c), thereby assesses the reactivity of described cell to described pharmacological agent.In one embodiment, described method further comprises the step to each repeating step (b)-(d) in the different in a large number secondary target genes.
In one embodiment, with described medicine the effect of the cell of the described cell type of not expressing a described siRNA is compared, described medicine strengthens the effect of one or more cells of expressing a described siRNA.In another embodiment, with described medicine the effect of the cell of the cell type of not expressing a described siRNA to be compared, described medicine weakens the effect of one or more cells of expressing a described siRNA.
In one embodiment, described composition comprises one or more inhibitor of described one or more secondary target genes.In a kind of preferred embodiment, described composition comprises fixed described one or more second target genes of target and makes one or more the 2nd siRNA of its silence.
In one embodiment, described one or more the 2nd siRNA comprise the different siRNA of k kind at least, and wherein said k is selected from 2,3,4,5,6 and 10.In one embodiment, the total siRNA concentration of the different siRNA of the described kind of k at least in the described reagent is the optimal concentration that makes described secondary target gene silencing, wherein said optimal concentration is such concentration, when this concentration of further increase, does not increase reticent level substantially.In one embodiment, described optimal concentration is such concentration, and when this concentration of further increase, the increase of reticent level is no more than more than 20%, more than 10% or 5%.In another embodiment, the concentration of each of the different siRNA of the described kind of k at least is approximately identical.In another embodiment, the different siRNA concentration separately of k kind difference each other is less than 50%, less than 20% or less than 10% at least.In another embodiment, do not have in the described reagent a kind of siRNA account for more than 80% of described total siRNA concentration of described different siRNA, more than 50% or more than 20%.In another embodiment, the concentration of at least a siRNA in the described reagent is more than 20% or more than 50% of described total siRNA concentration of the different siRNA of the described kind of k at least.In another embodiment, select the concentration of number He each siRNA of different siRNA in the described reagent, make described reagent cause any not as the silence of the gene of target less than 10%, less than 1%, less than 0.1% or less than 0.01%.
In some embodiments, described cell is the cancer cells type, and described primary target gene is p53.In preferred embodiments, described a large amount of secondary target gene comprise at least be selected from down the group heterogeneic number: 5,10,100,1000 with 5000 kinds of different genes.
In one embodiment, described medicine is the DNA disrupting agent, as is selected from the DNA disrupting agent of topoisomerase I inhibitor, topoisomerase II inhibitor, DNA wedding agent and ionizing rays.In a kind of specific embodiment, described DNA disrupting agent is selected from Zorubicin, camptothecine and cis-platinum.
4. accompanying drawing summary
Fig. 1 represents the mRNA silence of STK6 and the association between the growth-inhibiting phenotype.With six kinds of STK6 independent siRNA transfection HeLa cells.After transfection 24 hours, gather in the crops one group of cell and be used for RNA and separate, and determine the STK6mRNA level by adopting the TaqMan that measures (Assay onDemand) (Applied Biosciences) as required to analyze.Further another group cell (72 hours altogether) of incubation, and employing Alamar Blue is determined at assessment cell growth in three parts of holes.The rna level (X-axis) of each part and the value of cell growth (Y-axis) are carried out normalization method, so that the contrast of simulation transfection.Analyze for TaqMan, each data point representative is with the single rna sample (and normalizing to GUS) of three parts of mensuration; Variation between the repeated experiments usually<10%.For the determined value of growth measurement, each data point is represented the mean value of three replication values, the common departure of described measured value<20%.Solid line is represented 1: 1 relation of ideal between silence and the phenotype.
Fig. 2 represents that collaborative the causing death between STK6 and the KSP interacts.With luciferase (negative control) that increases concentration and the siRNA transfection HeLa cell of STK6 (last figure) or PTEN (figure below), and in three days Alamar Blue mensuration, test with respect to the growth that contrasts (luciferase processing).In the place of pointing out, cell is also used 25nM KSPi, (1S)-1-{[(2S)-4-(2, the 5-difluorophenyl)-2-phenyl-2,5-dihydro-1H-pyrroles-1-yl] carbonyl }-processing of 2-methyl propylamine; The EC50 of the HeLa cell of Ce Dinging is about 80nM under these conditions.Shown is the mean value ± SD (error post) of three replication values.
Fig. 3 has proved that the stably express of TP53shRNA makes the target gene silence effectively.Decide shRNA plasmid (pRS-p53) the transfection HCT116 cell of TP53 with target.What illustrate is wild-type (WT) cell and with the TP53mRNA level in two independent clonings (A5 and A11) of the cell of pRS-p53 stable transfection.Silence>95% (intermediary post) of TP53mRNA level in clone A5 and A11.In the instantaneous importing HCT116 of pRS-p53 cell, after transfection, reached about 80% silence (post on the right) in 24 hours.
After Fig. 4 is illustrated in the siRNA excess revolutions and dyes, express the silence of keeping mRNA by stable shRNA.(A) pRS-p53 does not influence the silence of the CHEK1 that siRNA causes, and vice versa.The set transient transfection of three kinds of siRNA of target being decided CHEK1 is to the HCT116 cell of WT and pRS-p53 stable transfection (clone A11).Analyze (being respectively left figure and right figure) by Taqman and measure CHEK1 and TP53mRNA level.(B) KNSL1siRNA that dyes of the excess revolutions silence that causes of competitive inhibition pRS-STK6 not.STK6 and KNSL1siRNA transient cotransfection in WT SW480 cell, and are dyed the KNSL1siRNA excess revolutions in the SW480 cell of pRS-STK6 stable transfection.By Taqman analysis to measure STK6mRNA level.For one group of post on the left side, use separately or the single KNSL1 siRNA (every kind 10nM) different with three kinds in a kind of STK6 siRNA (10nM) that uses together.KNSL1 siRNA suppresses the silence that STK6 siRNA causes changeably.For two groups of posts on the right, with 10 or 100nM with the competitor of KNSL1 siRNA as the STK6shRNA of stably express.
Fig. 5 has proved the siRNA library screening that does not exist under the DNA destructive condition, shows that having and do not have target decides good correlation between the cell of shRNA of p53.With pRS (the carrier is only arranged) cell (x axle) that the set excess revolutions of three kinds of siRNA is dyed, each of described three kinds of siRNA is one of fixed 800 kinds of genes of target all, and the relevant phenotype of test vector generation for testing IC; Measure pRS-p53 cell (y axle) in an identical manner.Substantial connection between two groups of data shows that the performance of siRNA set is not subjected to the influence of the existence of shRNA probably, shows that shRNA does not compete with siRNA.
The CHEK1 silence in the pRS-p53 cell of representing Fig. 6 has reduced the G2 outpost of the tax office and has stagnated (checkpoint arrest).Only use carrier (pRS) stable transfection A549 cell or dye the pRS-p53 cell with the set excess revolutions of three kinds of siRNA that contrast (luc, luciferase) siRNA or CHEK1.After transfection, added Zorubicin (200ng/ml) in 24 hours, and after adding Zorubicin 48 hours analysis of cells cyclic spectrums.Compare with the pRS cell, the TP53mRNA level in the pRS-p53 cell has reduced about 90%.
Fig. 7 has illustrated the evaluation to the gene of cis-platinum sensitivity.SiRNA with the about 800 kinds of people's genes of representative gathers the HeLa cell that (3siRNAs/ gene, total siRNA concentration is 100nM) transfection is grown on 384 orifice plates.After the transfection four hours, handle cell with independent substratum (or adding excipient) (medicine) or the substratum that adds the cis-platinum (Cis ,+medicine) of EC10 concentration.72 hours measurement cells are grown after being determined at transfection with Alamar Blue then, and are expressed as the growth % that measures in the hole with luciferase siRNA transfection.Each point is represented the mean number of 2-4 replication value.
Fig. 8 represents the comparison to the gene of different pharmacological agent sensitivities.By the siRNA transfection HeLa cell of use shown in Figure 1, and with independent substratum (or adding excipient) or add the substratum processing cell of Cis, Zorubicin (Dox) or the camptothecine (Campto) of EC10 concentration.The measurement cell is grown, and is expressed as the ratio of growth-medicine/growth+medicine.Dotted line is represented twice sensitization.Pointed out selected gene.
Fig. 9 A-9C represents that the silence of WEE1 makes the HeLa cell destroy responsive to Dox, Campto and Cis inductive DNA.Fig. 9 D-9I shows that the silence of WEE1 makes the p53-A549 cell destroy sensitivity to Dox, Campto and Cis inductive DNA, but it is responsive that the p53+A549 cell is destroyed described DNA.
Figure 10 A-10C shows that the silence of EPHB3 makes HeLa cell and p53-A549C7 destroy sensitivity to Dox, Campto and Cis inductive DNA, makes the p53+A549pRS cell lower to the degree of Dox, Campto and Cis inductive DNA destruction sensitivity.
Figure 11 A-11C shows that the STK6 silence makes HeLa cell and p53-A549C7 destroy sensitivity to Dox, Campto and Cis inductive DNA, makes the p53+A549pRS cell lower to the degree of Dox, Campto and Cis inductive DNA destruction sensitivity.
Figure 12 A-12C shows that the silence of BRCA1 makes HeLa cell and p53-A549C7 cell destroy responsive to Dox, Campto and Cis inductive DNA.It is responsive that the silence of BRCA also makes the p53+A549pRS cell destroy Cis inductive DNA with lower degree, but do not make the p53+A549pRS cell destroy responsive to Campto and Cis inductive DNA.
Figure 13 A-13B shows that the silence of BRCA2 makes HeLa cell and p53-A549C7 cell destroy responsive to Dox, Campto and Cis inductive DNA.It is responsive that Figure 13 C shows that the silence of BRCA makes the p53+A549pRS cell destroy Cis inductive DNA with lower degree, but do not make the p53+A549pRS cell destroy responsive to Campto and Cis inductive DNA.
Figure 14 A-14B shows that the silence of CHUK makes the HeLa cell destroy responsive to Dox, Campto and Cis inductive DNA.Figure 14 C shows that the silence of CHUK makes the p53-A549pRS cell destroy responsive to Campto and Cis inductive DNA.Figure 14 D shows that the silence of CHUK does not make the 53+A549pRS cell destroy responsive to Campto and Cis inductive DNA.
Figure 15 A-C shows the result of the reticent pair cell of CHEK1 to the influence of DNA destructive susceptibility.It is responsive that 15A:CHEK1 silence/inhibition makes the HeLa cell destroy DNA.It is responsive that 15B:CHEK1 silence/inhibition makes the p53-A549 cell destroy DNA.The 15C:CHEK1 silence does not make the HREP cell to the Zorubicin sensitivity.
Figure 16 represents that knock out (knockdown) of the PLK gene of siRNA mediation caused cell cycle arrest and apoptosis.
Figure 17 represents to screen the result to the gene of KSPi sensitivity.
Figure 18 represents to screen the result to the gene of taxol sensitivity.
Figure 19: the BRCA mixture has strengthened the cis-platinum activity., use cis-platinum (Y-axis) then or handle with the siRNA of about 2000 kinds of genes set (3siRNAs/ gene) transfection HeLa cell with 384 well format without cis-platinum (X-axis).Detect two kinds of different cis-platinum concentration, i.e. 100ng/ml (about EC10, left figure) or 400ng/ml (about EC50, right figure).After transfection 72 hours, measure the cell growth with Alamar Blue assay method.Diagonal lines is represented the consistence between twice processing (black line), or 2 times and 3 times of sensitization (being respectively fuchsin and red line) by cisplatin treated.
It is responsive that the silence of Figure 20: BRCA1 preferentially makes the TP53-cell that DNA is destroyed.With empty carrier (pRS, left figure) stable transfection A549 cell, or dye target with the siRNA excess revolutions of luciferase, BRCA1 or BRCA2 and decide the shRNA of TP53 (pRS-TP53, right figure), use DNA disrupting agent cisplatin treated then.After transfection 72 hours, measure the cell growth with Alamar Blue.
It is responsive that the reticent selectivity of Figure 21: BRCA1 makes the TP53-cell destroy DNA.With TP53 feminine gender (left column) or the positive (right row) the A549 cell that the siRNA transfection of luciferase (top line) or BRCA1 (end row) is mated, handle with DNA disrupting agent bleomycin then.After the transfection 72 hours, fixed cell, with the dyeing of iodate third ingot, and by the flow cytometry cell cycle distribution.Relative fluorescence with cell of 2N or 4N dna content is represented with arrow.The grid that marks with redness shows the number of inferior G1 (death) cell.
The result that Figure 22 represents proves that RAD51/ Zorubicin synergy is bigger in the TP53-cell.
5. detailed Description Of The Invention
The invention provides and adopt RNA to disturb identified gene or its product and a kind of reagent, for example interaction between the medicine is as causing death/the collaborative interactional method and composition that causes death.The term of Shi Yonging " gene product " comprises from the mRNA of genetic transcription with by the albumen of genes encoding herein.The present invention also provides and has utilized STK6 kinases (being also referred to as Aurora A kinases) and KSP (kinesin sample dynein is also referred to as KNSL1 or EG5) inhibitor (KSPi ' s) treatment method for cancer and composition.In this disclosure, use KSPi (1S)-1-{[(2S)-4-(2, the 5-difluorophenyl)-2-phenyl-2 usually, 5-dihydro-1H-pyrroles-1-yl] carbonyl }-2-methyl propylamine
(the PCT application PCT/US03/18482 referring to submitting on June 12nd, 2003 is incorporated herein by reference in full at this).Other KSPi ' s also can be used for the present invention.Can predict and use the method for described other KSPi ' s to be also included within the scope of the present invention.The present invention also provides the interaction that utilizes DNA to destroy between response gene and the DNA disrupting agent to treat method for cancer and composition.
5.1. disturb the interactional method of screening with RNA
The invention provides in the cell of identifying a kind of cell type and a kind of reagent, for example drug interaction, the method for for example regulating one or more genes of its effect.The interaction of the gene of Shi Yonging and a kind of reagent or another kind of gene herein comprises the interaction of gene and/or its product and this reagent or another kind of gene/gene product.For example, genes identified can be given the resistance of medicine or susceptibility,, reduces or strengthen the effect of medicine that is.Can adopt a large amount of siRNAs (each is one of a large amount of surely different genes of target all) to knock out a large amount of different genes (knocking out cell) in the cell of described cell type, and which kind of or which the generegulation cell in a large amount of different genes of determining to knock out is to the reaction of described reagent, thereby identifies described one or more genes.In one embodiment, a large amount of different cells (knocking out the library) that knock out of preparation knock out in the library each and knock out cell and all comprise the different genes that is for example knocked out by siRNA.In another embodiment, a large amount of different cells (knocking out the library) that knock out of preparation knock out in the library each and knock out cell and all comprise two or more different genes that for example knocked out by fixed heterogeneic shRNA of target and siRNA.In one embodiment, knock out the library and comprise a large amount of cells, wherein each is all expressed the siRNA that target is decided primary gene, and dyes with one or more siRNA excess revolutions of the fixed secondary gene of target.It will be apparent to one skilled in the art that also and can pass through other method, for example knock out cell by using target to decide the antisense molecule of gene or its product, ribozyme, antibody or the preparation of little organic or inorganic molecule.Can predict, any one in described other method and utilize the method for siRNA to be used singly or in combination is to prepare the library that knocks out of the present invention.Can use the method for any siRNA of being used for silence, keep the method for the reticent level that allows the adjusting target gene.Hereinafter 5.2 joints have been described operable several different methods.
In one embodiment, the siRNA that employing comprises a kind of a large amount of cells of cell type knocks out the library and implements method of the present invention, described every kind of cell all comprises one of a large amount of siRNA, among described a large amount of siRNA each all target is decided in the cell (promptly knocking out cell) one of a large amount of different genes and is made its silence (that is, knocking out).Any known method with the siRNA transfered cell can be used for this purpose.Preferably, prepare each cell in a large amount of cells, and keep independently, so that can independently study them.Use each cell in a large amount of cells of a kind of agent treated then, determine the effect of reagent this cell.Then with reagent to comprising by the effect of the cell of the gene of siRNA silence and this reagent to not comprising the cell of siRNA, i.e. the Normocellular effect of this cell type is compared.Identified and reacted on reagent and show the cell that knocks out of change.The described gene that knocks out in the cell by the siRNA silence that comprises is a gene of regulating the effect of described reagent.Preferably, a large amount of siRNA comprise target and decide at least 5 in the cell, 10,100 or 1000 kind of different gene and make its reticent siRNA.In a kind of preferred embodiment, described a large amount of siRNA targets are decided native gene and are made its silence.
In a kind of preferred embodiment, knock out the library and comprise a large amount of different cells that knock out, described cell has the identical gene that knocks out, and for example, it is homogenic and make its reticent siRNA that every kind of cell has different target phasings.Have the identical a large amount of different cells that knock out that knock out gene and can comprise at least 2,3,4,5,6 or 10 kind of different cell that knocks out, wherein each all comprises the siRNA that target knocks out the different zones of gene surely.In another kind of preferred embodiment, knock out the library and comprise a large amount of different cells that knock out, as, at least 2,3,4,5,6 or 10 kind, its at knock out in the library a large amount of heterogeneic each.In another kind of preferred embodiment, knock out the library and comprise a large amount of different cells that knock out, as, at least 2,3,4,5,6 or 10 kind, its at knock out in the library all heterogeneic each.
In another kind of preferred embodiment, knock out the library and comprise a large amount of different different cells that knock out that knock out gene that have, every kind different knocks out cell to have a target phasing homogenic and make two or more different siRNA of its silence.In preferred embodiments, each is different knocks out at least 2,3,4,5,6 or 10 kind of different siRNA that cell can comprise the isogenic different zones of target phasing.
In a kind of preferred embodiment, knock out the reaction assessment gene of cell and the interaction of reagent according to having a large amount of differences that knock out gene, for example, it is homogenic and make the different siRNA of its silence that every kind of cell has the target phasing.Utilize the reaction of a large amount of different siRNA, make it possible to determine that different siRNA are to the effect of target and non-target (referring to, the international application No.PCT/US2004/015439 of the Jackson that submitted on May 17th, 2004 etc. for example).
Compare with the normal cell of described cell type, reagent knocks out in the cell and can weaken the acting on of cell of described cell type, that is, gene knock out the effect that has weakened reagent.The described gene that is knocked out in described cell is considered to give the susceptibility to reagent.Therefore, in one embodiment, method of the present invention is used to identify one or more genes of giving the susceptibility of reagent.
Compare with the normal cell of described cell type, reagent knocks out in the cell and can strengthen the acting on of cell of described cell type.The described gene that is knocked out in described cell is considered to give the resistance to reagent.Therefore, in another embodiment, method of the present invention is used to identify one or more genes of giving the resistance of reagent.Enhancing to the reagent effect can be synergetic or collaborative.In one embodiment, the invention provides and can regulate and/or strengthen anticarcinogen in the cancer cells, as, one or more genes of the Growth Inhibition of KSP inhibitor in the cancer cells.
Method of the present invention can be used to assess a large amount of different reagent.For example, can be by the susceptibility of method assessment of the present invention to a large amount of different DNA disrupting agents of description in the 5.4.2 joint hereinafter.In a kind of preferred embodiment, assessment knocks out every kind of susceptibility that knocks out cell to each reagent in a large amount of different reagent in the library, to produce the reaction matrix of two dimension, wherein comprises every kind of reagent to every kind of observed value that knocks out the effect of cell.The intercept of gene axle under the specific gene index has produced the specific response curve that knocks out cell (wherein specific gene is knocked out) to different pharmaceutical.The intercept of the medicine axle under the certain drug condition has produced the gene response curve to medicine,, comprises that medicine knocks out the curve of observed value of the effect of cell to knocking out difference in the library that is.Table II A-IIC is the example to the gene response curve of cis-platinum (Table II A), camptothecine (Table II B) and Zorubicin (Table II C).
Method of the present invention can be used to identify the interaction between different genes, and this is by use regulating, and for example prevents or reinforcing gene expression and/or realized by the proteic active reagent of genes encoding.The example of described reagent includes, but not limited to target and decides the siRNA of gene or its product, antisense molecule, ribozyme, antibody and little organic or inorganic molecule.The fixed gene of described reagent place target is known as primary target.Described reagent can be used in combination with knocking out the library, regulates the gene of cell to the reaction of reagent to identify.Described primary target can be different from any one that knocks out a large amount of genes (secondary gene) of showing in the library.Therefore the gene that is accredited as the effect of regulating reagent is and the interactional gene of primary target.
In a kind of preferred embodiment, the invention provides the interactional method of using between the dual siRNA method evaluation different genes.In a kind of preferred embodiment, dual RNAi screening is to pass the excess revolutions of deciding the siRNA of secondary target gene with target and dye and realize by sending in the liptinite that uses the shRNA that destroys the primary target gene.This method provides coupling (isogenic) clone to (shRNA adds deduct), and does not cause the competition between shRNA and the siRNA.In the method, the recombinant vectors from instantaneous importing or stable integration to genome express short hairpin RNA (shRNA) (referring to, Paddison et al. for example, 2002, GenesDev 16:948-958; Sui et al., 2002, Proc Natl Acad Sci USA 99:5515-5520; Yu et al., 2002, Proc Natl Acad Sci USA 99:6047-6052; Miyagishi et al., 2002, Nat Biotechnol 20:497-500; Paul et al., 2002, Nat Biotechnol 20:505-508; Kwak et al., 2003, J Phannacol Sci 93:214-217; Brummelkamp et al., 2002, Science 296:550-553; Bodenetal., 2003, Nucleic Acids Res 31:5033-5038; Kawasaki et al., 2003, Nucleic Acids Res 31:700-707).Can express the siRNA that (through shRNA) destroys primary gene by any suitable carrier of coding shRNA.This carrier also can be encoded and be can be used for selecting the marker of cloning, and among the described clone carrier or its enough is partially integrated in the host genome, so that express shRNA.Any standard method well known in the art can be used for carrier sent and is delivered to cell.In one embodiment, by preparing the cell of expressing shRNA with the suitable cell of carrier-containing plasmid transfection.Can select cell by suitable marker then.Selected clone then detects being used to and knocks out.In a kind of preferred embodiment, the expression of shRNA is subjected to the control of inducible promoter, the feasible silence that can open its target gene when needed.The inducible expression of siRNA is specially adapted to the fixed necessary gene of target.
In one embodiment, the control of the promotor that the expression of shRNA is subjected to being regulated, described promotor makes it possible to regulate the reticent level of target gene.This makes it possible to screen specific cells, and the target gene in the described cell is partly knocked out.As used herein, " promotor of being regulated " is meant the promotor that can be activated when suitable inductor exists." inductor " is meant and anyly can be used for the promotor of being regulated by activation and the molecule of activated transcription.Inductor can be, but be not limited to peptide or polypeptide, hormone or organic molecule.Also can use the analogue of inductor, that is, activate the molecule of the promotor of being regulated as inductor.The activity level of the promotor that different analogue inductive are regulated can be different, thereby the adjusting of the activity level of feasible promotor of being regulated is more flexible.The promotor of being regulated in the carrier can be any Mammals transcriptional regulatory well known in the art system (referring to, for example, Gossen et al, 1995, Science 268:1766-1769; Lucas et al, 1992, Annu.Rev.Biochem.61:1131; Li et al., 1996, Cell 85:319-329; Saez et al., 2000, Proc.Natl.Acad.Sci.USA 97:14512-14517; With Pollock et al., 2000, Proc.Natl.Acad.Sci.USA 97:13221-13226).In preferred embodiments, the promotor of being regulated with dosage and/or analogue dependency mode.In one embodiment, by comprising the method for the concentration that the concentration adjustment of inductor can be reacted to the promotor of being regulated, the activity level of the promotor of being regulated is adjusted to the level that needs.Can be according to the reticent level of required target gene, determine the desired level of the promoter activity of being regulated that obtains by the inductor of using specific concentrations.
In one embodiment, use the gene expression system that tsiklomitsin regulates (referring to, Gossen et al for example, 1995, Science 268:1766-1769; U.S. Patent No. 6,004,941).The system that tet regulates uses the composition of procaryotic tet repressor/operon/inductor system to regulate genetic expression in the eukaryotic cell.Like this, the invention provides the expression of using the tet regulation system to regulate the shRNA that is connected with one or more tet operon sequence.This method comprises the carrier transfered cell with the fusion rotein of coding activated transcription.Fusion rotein is included in tsiklomitsin or tetracycline analogue and exists down and tet operon sequence bonded first polypeptide, and described tsiklomitsin or tetracycline analogue are connected with the second polypeptide operability of transcribing in the activating cells.By regulating the concentration of tsiklomitsin or tetracycline analogue, regulate the expression of the shRNA of tet operon connection.
In other embodiments, can use the gene expression system that moulting hormone regulates (referring to, for example, Saez et al., 2000, Proc.Natl.Acad.Sci.USA 97:14512-14517) or the gene expression system regulated of MMTV glucocorticoid responsive element (referring to, for example, Lucas et al, 1992, the Annu.Rev.Biochem.61:1131) expression of adjusting shRNA.
In one embodiment, use coding tetracycline resistance markers and driving pRETRO-SUPER (pRS) carrier from H1 (RNA Pol III) promoter expression shRNA.Can prepare the pRS-shRNA plasmid by standard method well known in the art.In one embodiment, pRS-shRNA is from library plasmid set deconvolution, and this is by gathering transform bacteria with this, and seeks the clone who only contains interested plasmid.Preferably, 19 aggressiveness siRNA sequences are used with the suitable forward and the reverse primer that are used for sequence-specific PCR.Identify plasmid by sequence-specific PCR, and confirm by order-checking.By express the cell of shRNA with the suitable cell preparation of pRS-shRNA plasmid transfection.By suitable marker, select cell as tetracycline, and keep up to colony obvious.Selected clone then, and detect being used to and knock out.
In another embodiment,, express shRNA as pRS-shRNA by plasmid.By knocking out that pRS-shRNA carries out, can be by realizing with Lipofectamine 2000 (Invitrogen) transfectional cell.
In a kind of preferred embodiment, prepare the clone (+/-primary target gene) of coupling by the stable clone of selecting to contain sky pRS carrier or pRS-shRNA.
The shRNA primary target clone's of employing preparation cell carries out the silence of secondary target gene then.Can adopt any known RNA interference method finish the silence of secondary target gene (referring to, for example 5.2 the joint).For example, can be with the plasmid transfection of siRNA and/or coding shRNA, so that the secondary target gene silencing.In one embodiment, the shRNA primary target clone's of preparation cell is dyed in one or more siRNA excess revolutions of deciding the secondary target gene with target.In one embodiment, target is decided one or more siRNA direct transfection of secondary gene in cell.In another embodiment, adopt one or more suitable plasmids, through shRNA target is decided one or more siRNA transfections of secondary gene in cell.Can be after transfection 24 hours results RNA, and knock out by the TaqMan analysis and evaluation.In a kind of preferred embodiment, will contain target and decide the siRNA set of the different siRNA of the kind of k at least of the different sequence areas of secondary target gene (k=2,3,4,5,6 or 10) and be used for the excess revolutions transfect cell.In another kind of preferred embodiment, the siRNA set that will contain the different siRNA of the kind of k at least (k=2,3,4,5,6 or 10) of fixed two or more the different secondary target genes of target is used for transfectional cell.
In a kind of preferred embodiment, total siRNA concentration of the set approximately concentration with the single siRNA that uses separately is identical, for example 100nM.Preferably, the total concn of siRNA set is the optimal concentration that is used to make as the target gene silence of target.Optimal concentration is such concentration, when this concentration of further increase, does not increase reticent level substantially.In one embodiment, optimal concentration is such concentration, and when this concentration of further increase, the increase of reticent level is no more than 5%, 10% or 20%.In a kind of preferred embodiment, select the composition of set, the concentration that comprises number with each the different siRNA of different siRNA in the set, make the set of siRNA cause any not as the silence of the gene of target less than 30%, 20%, 10% or 5%, 1%, 0.1 or 0.01%.In another kind of preferred embodiment, the concentration of the every kind of different siRNA that comprises in the set of different siRNA is approximately identical.In another kind of preferred embodiment, the concentration difference each other of the different siRNA that comprises in the set is less than 5%, 10%, 20% or 50%.In a kind of preferred embodiment, at least a siRNA in the set of different siRNA accounts for more than 90%, 80%, 70%, 50% or 20% of total siRNA concentration of set.In another kind of preferred embodiment, there is not a kind of siRNA to account for more than 90%, 80%, 70%, 50% or 20% of total siRNA concentration of different siRNA in the set of different siRNA.Optimal concentration when in other embodiments, the concentration of each siRNA in the set all is lower than independent the use.In a kind of preferred embodiment, the concentration of the siRNA that each in the set is different is lower than at least 30%, 50%, 75%, 80%, 85%, 90 or 95% siRNA concentration of the gene silencing that effectively reaches when not having other siRNA or not existing other to be designed for the siRNA that makes gene silencing.In another kind of preferred embodiment, the gene silencing that the concentration of the different siRNA of each in the set causes is lower than 30%, 20%, 10% or 5% of gene silencing when not having other siRNA or not existing other to be designed for the siRNA that makes gene silencing.In a kind of preferred embodiment, 30%, 20%, 10% or 5% of the target gene silence that the gene silencing that the concentration of every kind of siRNA causes causes when being lower than independent the use, and a large amount of siRNA causes at least 80 or 90% of target gene silence.
In one embodiment, the invention provides and be used to identify the method that shows with collaborative interactional one or more genes that cause death of primary target gene.In the method, the reagent that is used as the inhibitor of the primary target gene in the cell type screens knocking out the library.Therefore, being accredited as the gene of the effect that strengthens reagent, is to have the collaborative interactional gene that causes death with primary target.In a kind of preferred embodiment, described reagent is that target is decided primary target and made its reticent siRNA.
The method of determining the effect of reagent pair cell depends on specific function to be assessed.For example, if reagent is cancer therapy drug, and effect to be assessed is the growth-inhibiting effect of medicine, then can use MTT to measure or alamarBlue measures (referring to, 5.2 joints for example).Those skilled in the art personnel can select method well known in the art based on specific function to be assessed.
In another embodiment, the invention provides and determine the effect of a kind of reagent to the growth of specific cells, the primary target gene of described specific cells and secondary target gene silencing.In a kind of preferred embodiment, according to the clone of preparation mentioned above coupling (+/-primary target gene).Two kinds of clones are dyed in one or more siRNA excess revolutions of deciding the secondary target gene with contrast siRNA (as luciferase) or target then.Under the condition that exposes or be not exposed to described reagent, check the cell cycle spectrum.Can carry out cell cycle analysis (5.2 joints vide infra) with standard method well known in the art.In one embodiment, the supernatant liquor with each hole merges with the cell of gathering in the crops by tryptic digestion.Then with the proper speed centrifugal mixture.Use the 70% ice-cold alcohol time period that cell fixation is suitable then, 30 minutes according to appointment.Can wash the fixed cell once with PBS, resuspended then, for example be resuspended in 0.5ml and contain iodate third ingot (among the PBS of 10 micrograms/ml) and RNA enzyme A (1mg/ml), in suitable temperature, as 37 ℃ of time periods that following incubation is suitable, as 30 minutes.Carry out flow cytometry with flow cytometer.In one embodiment, measure necrocytosis with inferior G1 cell mass.The increase of the G1 cell mass in the cell of primary target gene and secondary target gene silencing shows the collaborative lethal effect of primary and secondary target gene in the presence of described reagent.
In a kind of particular, the invention provides the method that is used for identified gene, knocking out of described gene strengthens the growth-inhibiting effect of KSP inhibitor to tumour cell.In one embodiment, with this method identified gene, knocking out in inferior optimal concentration of described gene promptly is lower than the following growth of tumour cell that suppresses of KSPi existence of the concentration of EC10.In one embodiment, each the siRNA of 3 kinds of siRNA contain in the fixed following 11 kinds of genes of target of preparation and use knocks out the library: CDC20, ROCK2, TTK, FZR1, BUB1, BUB3, BUB1B, MAD1L1, MAD2L1, DNCH1 and STK6 (referring to table 1).KSPi in<EC10 concentration (25nM), i.e. (1S)-1-{[(2S)-4-(2, the 5-difluorophenyl)-2-phenyl-2,5-dihydro-1H-pyrroles-1-yl] carbonyl-2-methyl propylamine (referring to the PCT application PCT/US03/18482 that submitted on June 12nd, 2003) exist or non-existent condition under among these siRNA each is imported HeLa cell, and the reaction of definite cell.The siRNA of a kind of STK6 (STK6-1) shows KSPi and has following remarkable inhibition to growth of tumour cell.
Further check growth inhibitory activity with three kinds of STK6 extra siRNA, and assess all 6 kinds of siRNA and induce the reticent and growth inhibiting ability of STK6.In different siRNA, between reticent level of STK6 and growth-inhibiting, there is good dependency (Fig. 1).This dependency shows that growth-inhibiting is because target activity (that is, STK6 destroys).(1S)-the 1-{[(2S)-4-(2 that in the PCT application PCT/US03/18482 that submitted on June 12nd, 2003, describes then, the 5-difluorophenyl)-2-phenyl-2,5-dihydro-1H-pyrroles-1-yl] carbonyl-2-methyl propylamine exist or non-existent condition under, decide contrast (negative control) the titration STK6-1 (Fig. 2) of luciferase with target.The interpolation of KSPi has been moved to the left about 5-10 doubly with the STK6-1 dose response curve.This concentration of KSPi does not have to strengthen the effect of the cell growth that is caused by luciferase siRNA.On the contrary, the dose response curve of target being decided the siRNA of PTEN is not moved by KSPi, and described PTEN has similar effect to the STK-6 cell growth.The siRNA that other target is decided STK6 also strengthens the effect of KSPi cell growth.Thus, the destruction of STK6 has strengthened the effect of KSPi cell growth.The research of the combination of the siRNA by adopting STK6 and KSP has obtained the further support (table 1) to this, and described combination table reveals than arbitrary independent stronger growth inhibitory activity of siRNA.Do not influence cell growth owing to be used for KSPi concentration self of these experiments, active effect shows as collaborative, rather than synergetic KSPi to STK6siRNA.
In another kind of particular, the invention provides the method that is used for determining the collaborative lethal effect between p53 and the CHEK1.The stable clone that has prepared the p53 gene silencing.PRS-TP53 1026 shRNA plasmids are from the library plasmid set deconvolution of TP53, and this is by gathering transform bacteria with this, and seeks the clone who only contains interested plasmid.Employed sequence is as follows: pRS-p53 1,026 19 aggressiveness sequence: GACTCCAGTGGTAATCTAC (SEQ ID NO:43); The primer of sequence-specific PCR: forward: GTAGATTACCACTGGAGTC (SEQ ID NO:44), oppositely: CCCTTGAACCTCCTCGTTCGACC (SEQ ID NO:45).Identify plasmid by sequence-specific PCR, and confirm by order-checking.By with FuGENE 6 (Roche) the transfection HCT116 cell that has the pRS-TP531026 plasmid, prepare stable p 53 clones.After 48 hours cell is assigned in the 10cm culture dish that is added with the 1ug/ml tetracycline, keeps up to colony obvious (5-7 days).The clone is selected 96 orifice plates, in the 1ug/ml tetracycline, keep, and use TP53 and hGUS Pre-Developed to measure reagent (AppliedBiosystems), knock out by the TaqMan check.In order to measure instantaneous knocking out,, after 24 hours, gather in the crops RNA with Lipofectamine 2000 (Invitrogen) transfection HCT116 cell by the pRS-TP531026 plasmid.By TaqMan assessment TP53 transcript level.
Derive from colon tumor cell and be a plurality of tetracycline resistance TP53shRNA clones' (pRS-p53) of HCT116 the target silence (being defined as 50%-96%) of analysis revealed different levels by TaqMan.Fig. 3 shows the TP53 expression level among clone A5 and the A11, and described clone shows the silence of highest level.TP53 silence among these clones has surpassed by transient transfection pRS-p53 to be sent and has been delivered to 24 hours observed silences (Fig. 3) behind the HCT116 cell.Transfection efficiency may the validity of restricted T P53shRNA in transient transfection.Perhaps, the cell with the reticent level of higher TP53 has obtained growth vigor in the clonal growth process.Adopt target to decide the shRNA (pRS-STK6:pRS-STK6 2,178 19 aggressiveness sequence: CATTGGAGTCATAGCATGT (SEQ ID NO:46)) of STK6, also observed the silence of the certain limit in the stable clone.But, these clones do not reach with TP53 clone in the silence of observed the same high level, its silence also is no more than the silence that obtains in instantaneous measurement.This may represent the selection of anti-high-level STK6 silence, because STK6 is the necessary gene of the growth of tumour cell in the culture.
In order to check the TP53 silence among the HCT116 clone whether to compete, dye this clone's cell with the set excess revolutions of CHEK1 specific siRNA with siRNA.The CHK1 set contains following three kinds of siRNA:CUGAAGAAGCAGUCGCAGUTT (SEQ ID NO:99); AUCGAUUCUGCUCCUCUAGTT (SEQID NO:98); And UGCCUGAAAGAGACUUGUGTT (SEQ ID NO:100).Find the reticent activity of the siRNA that the competitive TP53 of reduction of this siRNA set target is fixed.Show siRNA with 10nM or 100nM Oligofectamine (Invitrogen) transfection.For the CHK1 set,, send altogether and pass 100nM with three kinds of siRNA of 33.3nM transfection simultaneously.24 hours results RNA after transfection, and use CHK1 or TP53 and hGUS Pre-Developed to measure reagent (AppliedBiosystems), knock out by the TaqMan analysis and evaluation.Shown in Fig. 4 A, shRNA and siRNA gather the not silence of every kind of other target of competitive inhibition.Measure the inhibition that the known competitive siRNA by the shRNA of the siRNA of the transient transfection of identical sequence or stably express causes then.Shown in Fig. 4 B, three kinds of independent targets are decided KNSL1 (KNSLI210:GACCUGUGCCUUUUAGAGATT (SEQ ID NO:47); KNSLI211:GACUUCAUUGACAGUGGCCTT (SEQ ID NO:48); KNSLI212:AAAGGACAACUGCAGCUACTT (SEQ ID NO:49)) siRNA competitive inhibition is decided the silence (post on the left side) that the siRNA of the cotransfection of STK6 reaches by target.On the contrary, the influence that the silence that homology STK6shRNA causes in the clone of stable transfection is not dyed by the excess revolutions of KNSL1siRNA is even when adding competitor siRNA with high 10 times concentration also be so the post of (middle and the right).These experiments show, almost not competition between the siRNA of the shRNA of stably express and transient transfection.Observations during two kinds of different siRNA of that this competes each other with transfection together, fixed different mRNA of target is opposite, wherein effectively reduces the efficient of one or both siRNA of use.With the siRNA set transient transfection pRS and the pRS-p53HCT116 cell (embodiment 3 vide infra) of about 800 kinds of genes, and the effect of measuring cell growth by Alamar Blue assay method.Observed reaction much at one, do not shown reticent competitive inhibition the set of about 800 kinds of siRNA described in pRS cell and the pRS-p53 cell.
Subsequently, the excess revolutions that assessment CHEK1 siRNA is integrated in the cell of stably express TP53shRNA is dyed, to determine whether to use it for the genetic interaction (SL) between these molecules of research.By select to contain the clone that the A549 lung cancer cell line that free pRS carrier or pRS-p53 are arranged prepares coupling+/-TP53 expresses.A kind of cell in back shows>90% TP53mRNA silence.Then with contrast luciferase siRNA (luc, 100nM) or CHEK1siRNA set (100nM altogether; Each 33nM of 3 kinds of siRNA) two kinds of clones are dyed in excess revolutions, are exposing or be not exposed to DNA disrupting agent Zorubicin (Dox, their cell cycle spectrum of check under situation Fig. 5).Under the situation that lacks Dox, the cell cycle of pRS-p53 cell spectrum obviously is not different from the cell cycle spectrum of pRS cell.The transient transfection of CHEK1siRNA does not have influence not exist the cell cycle under the Dox situation to compose yet.But under the situation that Dox exists, as desired to the cell of expressing functional TP53, the pRS cells transfected has shown G1 and G2/M stagnates.The excess revolutions of CHEK1siRNA is dyed and has been caused crossing the G2 outpost of the tax office, and increases at the cell number of G1 blocking-up.Because cell keeps the TP53 function, they were stagnated and are not got back to the S phase in the G1 phase.
On the contrary, the pRS-p53 cell has lost the ability of stagnating at G1, and principal reaction handles and stagnate in the G2 phase in Dox, and this is consistent in the effect at the G1 outpost of the tax office with TP53.Cell cycle spectrum by the existing pRS-p53 cell of lucsiRNA excess revolutions hair dyeing does not change (Fig. 5).LucsiRNA even the part that does not cause TP53 to react are recovered (and the corresponding increase at G1 peak), show that this siRNA does not almost have to produce the competitive inhibition of and phenotype reticent to TP53.Therefore, estimate not exist CHEK1 siRNA to gather the competitive inhibition of the TP53 silence that causes.In fact, react on Dox and handle, revealed remarkable change with the pRS-p53 cell of CHEK1 transient transfection at their cell cycle stave, the cell proportion in the S phase significantly increases, and has the DNA that inferior G1 (dead cell) measures.HCT116 cell kind at pRS and pRS-p53 stable transfection has also been observed similar discovery.Therefore, destroying in the time of by the G2 outpost of the tax office of the G1 outpost of the tax office of TP53 mediation and CHEK1 mediation, is lethal to the TP53-tumour cell, but is not lethal to the TP53+ tumour cell.
In another embodiment, the invention provides a member who determines p53 and BRCC mixture, as the method for the collaborative lethal effect between BRCA1, BRCA2, BARD1 and the RAD51.In this embodiment, used a pair of TP53 positive and the negative cells of coupling of decide short hairpin RNA (shRNA) generation of TP53 by the stably express target.Dye TP53 positive or negative cell with the siRNA of BRCA1 or BRCA2 set excess revolutions, use cisplatin treated, and with Alamar Blue analysis of cells grow (Figure 20).When with BRCA1 or BRCA2siRNAs (the about 0.1nM of IC50) transfection, sensitivity was about 10 times when the TP53 negative cells was used contrast siRNA (luciferase, the about 1nM of IC50) transfection to the cis-platinum ratio.In lower cis-platinum concentration, BRCA1 or BRCA2 destroy the back to the sensibilized of cis-platinum even more remarkable.After BRCA1 or BRCA2 destroyed, the TP53 positive cell was to cis-platinum more insensitive (the about 0.4nM of IC50).In this is analyzed, BRCA1 or BRCA2 destroy the back to the sensibilized of cis-platinum on the order of magnitude with sensibilized similar (data not shown goes out) after CHEK1 destruction.Also can use cell cycle analysis, research BRCA1 and BRCA2 destroy the sensibilized of back to the DNA disrupting agent.The TP53 positive and negative cells are dyed in siRNA set excess revolutions with BRCA1 or BRCA2, with a kind of processing in some DNA disrupting agents (cis-platinum, camptothecine, Zorubicin and bleomycin), and by flow cytometry cell cycle destruction.In all cases, the TP53 negative cells BRCA1 and BRCA2 destroy the back than in the luciferase cells transfected to DNA disrupting agent more responsive (data not shown goes out).These cells were shown in Figure 21 to the reaction of bleomycin after BRCA1 destroyed.BRCA1 destroys after handling the TP53 negative cells with bleomycin, has caused more inferior G1 cell (dead cell) than handling the TP53 positive cell.Described result shows that the cell that lacks TP53 destroys the back at BRCA1 and destroys more responsive to DNA.
The clone of using can be positive A549 cell of HeLa cell, TP53 or the negative A549 cell of TP53.In one embodiment, decide the short hairpin RNA (shRNA) of TP53 by the stable transfection target, prepared the positive and negative cells of the TP53 of coupling to (monthlyhighlt highlight, Nov.2003).With the siRNA of 100nM (every kind of siRNA 33nM) set (set of three kinds of siRNA of each gene) transfectional cell, or with the single siRNA transfectional cell of 100nM.Following siRNA:Luc contrast, BRCA1, BRCA2 and BARD1 set have been used.Handle cells transfected with the DNA disrupting agent of various concentration then.The concentration of the every kind of reagent that uses in the cell cycle analysis is as follows: for the HeLa cell, use Zorubicin (10nM), camptothecine (6nM), cis-platinum (400ng/ml), ametycin (40nM), bleomycin (100ng/ml); For other cell, use Zorubicin (200nM), camptothecine (200nM), cis-platinum (2ug/ml) ametycin (400nM), bleomycin (5ug/ml).
In one embodiment, be performed as follows the siRNA transfection: in transfection the day before yesterday, the clone that 2000 (or 100) microlitre is selected, as at DMEM/10% foetal calf serum (Invitrogen, Carlsbad, CA) (ATCC Cat.No.CCL-2) is planted in the tissue culturing plate of 6 holes (or 96 holes) with 45,000 (or 2000) cells/well to grow to the about 90% cervical cancer HeLa cell that converges in.For each transfection, with 70 microlitre OptiMEM (Invitrogen) with (Dharmacon, Lafayette CO) mix from 5 microlitre siRNA of 20 micromole's liquid storages.For each transfection, 20 microlitre OptiMEM are mixed with 5 microlitre Oligofectamine reagent (Invitrogen), and incubation 5 minutes at room temperature.Then 25 microlitre OptiMEM/Oligofectamine mixtures are mixed with 75 microlitre OptiMEM/siRNA mixtures, and at room temperature incubation 15-20 minute.100 (or 10) microlitre transfection mixture is distributed in each hole of 6 holes (or 96 holes) plate, and under the condition of 37 ℃ and 5%CO2 incubation 4 hours.
After 4 hours, the DMEM/10% foetal calf serum that contains or do not contain the DNA disrupting agent in 100 microlitres/hole is added each hole, to reach the final concentration of every kind of reagent mentioned above.With plate incubation 68 hours again under the condition of 37 ℃ and 5%CO2.Analysis is from the cell cycle spectrum of the sample of 6 orifice plates, with the cell growth of Alamar Blue assay method analysis from the sample of 96 orifice plates.
For cell cycle analysis, will be from the supernatant liquor and the cytomixis of gathering in the crops by tryptic digestion in each hole.Then with 1200rpm with centrifugal 5 minutes of mixture.With 70% ice-cold ethanol with about 30 minutes of cell fixation.With PBS once, be suspended in 0.5ml again and contain iodate third ingot (among the PBS of 10 micrograms/ml) and RNA enzyme A (1mg/ml), and 37 ℃ of following incubations 30 minutes with the fixed cell washing.(Becton Dickinson) carries out flow cytometry with the FACSCalibur flow cytometer, and with FlowJo software (TreeStar, Inc) analytical data.Measure necrocytosis with inferior G1 cell mass.If (siRNA+DMSO) summation of the inferior G1 cell mass of sample and (Luc+ medicine) sample is greater than the inferior G1 cell mass of (siRNA+ medicine) sample, we determine that the siRNA silence is to the sensitization of DNA destructive.
Analyze for Alamar Blue, remove the substratum of 96 orifice plates, add the 100uL/ hole contain 10% (vol/vol) alamar Blue reagent (BioSource International, Inc) and the perfect medium of 1 percent volume 1M Hepes damping fluid tissue culture reagent.Then with plate at 37 ℃ of following incubation 1-4 hours, by exciting and detect emission at 590nm and measure fluorescence at 544nm with SPECTRAMax Gemini-Xs spectrofluorimeter (Molceular Devices).Background (acellular) is proofreaied and correct fluorescent signal.Cell response (survival) under the DNA disrupting agent exists is measured as the per-cent of the control cells growth that does not exist under the DNA disrupting agent condition.
5.2. be used for the method and composition of RNA interference and raji cell assay Raji
The standard method of any gene silencing can be used for the present invention (referring to, for example, Guo etal., 1995, Cell 81:611-620; Fire et al., 1998, Nature 391:806-811; Grant, 1999, Cell 96:303-306; Tabara et al., 1999, Cell 99:123-132; Zamore et al., 2000, Cell 101:25-33; Bass, 2000, Cell 101:235-238; Petcherski et al., 2000, Nature 405:364-368; Elbashir et al., Nature 411:494-498; Paddison et al., Proc.Natl.Acad.Sci.USA 99:1443-1448).Can according to method well known in the art design target decide gene siRNA (referring to, the U.S. Provisional Patent Application No.60/572 of the Jackson that submitted on May 17th, 2004 etc. for example, 314, with Elbashir et al., 2002, Methods 26:199-213, at this complete content of introducing each piece in full as a reference).
Only can use with target gene have the partial sequence homology siRNA (referring to, the international application No.PCT/US2004/015439 of the Jackson that example was submitted on May 17th, 2004 etc. introduces its complete content as a reference in full at this).In one embodiment, use the sense strand continuous nucleotide sequence comprise 11-18 the Nucleotide identical with the sequence of genetic transcription thing siRNA (but this siRNA not with transcript in any sequence have the total length homology) make gene silencing.Preferably, the continuous nucleotide sequence is the central section of siRNA molecule.Continuous nucleotide sequence in the central section can be not since 3 ' any one section successive nucleotide sequence of holding among the siRNA.For example, the continuous nucleotide sequence of 11 Nucleotide can be nucleotide sequence 2-12,3-13,4-14,5-15,6-16,7-17,8-18 or 9-19.In a kind of preferred embodiment, the length of continuous nucleotide sequence is 11-16,11-15,14-15,11,12 or 13 Nucleotide.
In another embodiment, use 3 ' the sense strand continuous nucleotide sequence comprise 9-18 the Nucleotide identical with the sequence of genetic transcription thing siRNA (but this siRNA not with transcript in any continuous sequence have the total length homology) make gene silencing.In this application, the sequence of 3 ' 9-18 Nucleotide is one section successive Nucleotide, and it is since first pair of base, that is, it does not comprise 3 ' overhang of two bases.Therefore, when pointing out that specific nucleotide sequence is positioned at the 3 ' end of siRNA, do not consider 2 base overhangs.In preferred embodiments, the length of continuous nucleotide sequence is 9-16,9-15,9-12,11,10 or 9 Nucleotide.
Can carry out RNA with any method well known in the art disturbs.In one embodiment, by siRNA induced gene silence is provided to cell, the product of simulation nickase cutting (referring to, Elbashir et al. for example, 2001, Nature 411,494-498; Elbashir et al., 2001, Genes Dev.15,188-200 introduces all documents as a reference in full at this).Synthetic siRNA duplex has kept the associating ability with RISC, and instructs the silence of mRNA transcript.SiRNA can chemosynthesis, or derives from the cutting of the double-stranded RNA that is undertaken by the reorganization nickase.Can adopt standard method well known in the art, use the siRNA transfectional cell.
In one embodiment, be performed as follows the siRNA transfection: in transfection the day before yesterday, the cell that 100 microlitres are selected, as at DMEM/10% foetal calf serum (Invitrogen, Carlsbad, (ATCC Cat.No.CCL-2) is planted in (Corning in the 96 hole tissue culturing plates with 1500 cells/well to grow to the about 90% cervical cancer HeLa cell that converges in CA), Coming, NY).For each transfection, (Dharmacon Denver) mixes with siRNA from 5 microlitre serial dilutions of 20 micromole's liquid storages with 85 microlitre OptiMEM (Invitrogen).For each transfection, 5 microlitre OptiMEM are mixed with 5 microlitre Oligofectamine reagent (Invitrogen), and incubation 5 minutes at room temperature.Then 10 microlitre OptiMEM/Oligofectamine mixtures and OptiMEM/siRNA mixture are assigned in each test tube, mix, and at room temperature incubation 15-20 minute.10 microlitre transfection mixtures are distributed in each hole of 96 orifice plates, and under the condition of 37 ℃ and 5%CO2 incubation 4 hours.
The another kind of method that is used for gene silencing is to import shRNA, promptly short hairpin RNA (referring to, Paddison et al. for example, 2002, Genes Dev.16,948-958; Brummelkampet al., 2002, Science 296,550-553; Sui, G.et al.2002, Proc.Natl.Acad.Sci.USA 99,5515-5520 introduces all above-mentioned documents as a reference in full at this).In the method, from expressing the siRNA that needs as plasmid (or virus) to form hairpin structure with inverted repeats of the ring-shaped sequence that interleaves.The rna transcription thing that contains hairpin structure that obtains by nickase processing is used to carry out reticent siRNA with preparation subsequently.Can in cell, make it possible in vitro and in vivo based on the shRNA of plasmid by stably express, for example in Mammals, carry out long-term gene silencing (referring to, McCaffrey et al.2002, Nature418,38-39; Xia et al., 2002, Nat.Biotech.20,1006-1010; Lewis et al., 2002, Nat.Genetics 32,107-108; Rubinson et al., 2003, Nat.Genetics33,401-406; Tiscornia et al., 2003, Proc.Natl.Acad.Sci.USA 100,1844-1848 introduces all above-mentioned documents as a reference in full at this).Like this, in one embodiment, use shRNA based on plasmid.
In a kind of preferred embodiment, will from the instantaneous importing of the shRNA that recombinant vectors is expressed or stable integration to genome (referring to, Paddison et al. for example, 2002, Genes Dev16:948-958; Sui et al., 2002, Proc Natl Acad Sci USA 99:5515-5520; Yu et al., 2002, Proc Natl Acad Sci U SA 99:6047-6052; Miyagishi etal., 2002, Nat Biotechnol 20:497-500; Paul et al., 2002, Nat Biotechnol20:505-508; Kwak et al., 2003, J Phamacol Sci 93:214-217; Brummelkamp et al., 2002, Science 296:550-553; Boden et al., 2003, Nucleic Acids Res 31:5033-5038; Kawasaki et al., 2003, Nucleic AcidsRes 31:700-707).Can express the siRNA that destroys target gene by the carrier (through shRNA) of any suitable coding shRNA.Carrier also can be encoded and is used to select the marker of specific cloning, and carrier or its enough have been partially integrated in the host genome among the described clone, so that express shRNA.Can carrier be sent with any method well known in the art and be delivered in the cell.In one embodiment, by preparing the cell of expressing shRNA with the suitable cell of plasmid transfection that comprises carrier.Can select cell by suitable marker then.Selected clone then, testing knocks out being used to.In a kind of preferred embodiment, the expression of shRNA is under the control of inducible promoter, can open the silence of its target gene when needing with box lunch.The inducible expression of siRNA is specially adapted to the fixed necessary gene of target.
In one embodiment, the expression of shRNA is under the control of the promotor of being regulated, and described promotor makes it possible to regulate the reticent level of target gene.This makes it possible to screen the cell that target gene is partly knocked out." promotor of being regulated " of Shi Yonging is meant the promotor that can be activated when having suitable inductor herein." inductor " can be anyly can be used to the promotor of being regulated by activation and the molecule of activated transcription.Inductor can be, but be not limited to peptide or polypeptide, hormone or organic molecule.Also can use the analogue of inductor, that is, and the same molecule that activates the promotor of being regulated with inductor.The activity level of the promotor of being regulated by different analogue inductive can be different, make that like this activity level adjusting of the promotor of being regulated is more flexible.The promotor of being regulated in the carrier can be any Mammals transcriptional regulatory well known in the art system (referring to, Gossen et al for example, 1995, Science 268:1766-1769; Lucas et al, 1992, Annu.Rev.Biochem.61:1131; Li et al., 1996, Cell 85:319-329; Saez et al., 2000, Proc.Natl.Acad.Sci.USA97:14512-14517; With Pollock et al., 2000, Proc.Natl.Acad.Sci.USA97:13221-13226).In preferred embodiments, the promotor of being regulated with dosage and/or analogue dependency mode.In one embodiment, by comprising the method for the concentration that the concentration adjustment of inductor is responded to the promotor of being regulated, the activity level of the promotor of being regulated is adjusted to the level that needs.Can be based on the reticent level of the needs of target gene, determine activity level by the needs of using the promotor of being regulated that the specific concentrations inductor obtains.
In one embodiment, use the gene expression system that tsiklomitsin regulates (referring to, Gossen et al for example, 1995, Science 268:1766-1769; U.S. Patent No. 6,004,941).The system that tet regulates uses the composition of procaryotic tet repressor/operon/inductor system to regulate genetic expression in the eukaryotic cell.Like this, the invention provides the expression of using the tet regulation system to regulate the shRNA that is connected with one or more tet operon sequence.This method comprises the carrier transfered cell with the fusion rotein of coding activated transcription.Fusion rotein is included in tsiklomitsin or tetracycline analogue and exists down and tet operon sequence bonded first polypeptide, and described tsiklomitsin or tetracycline analogue are connected with the second polypeptide operability of transcribing in the activating cells.By regulating the concentration of tsiklomitsin or tetracycline analogue, regulate the expression of the shRNA of tet operon connection.
In other embodiments, can use the gene expression system that moulting hormone regulates (referring to, for example, Saez et al., 2000, Proc.Natl.Acad.Sci.USA 97:14512-14517) or the gene expression system regulated of MMTV glucocorticoid responsive element (referring to, for example, Lucas et al, 1992, the Annu.Rev.Biochem.61:1131) expression of adjusting shRNA.
In one embodiment, use coding tetracycline resistance markers and driving pRETRO-SUPER (pRS) carrier from H1 (RNA Pol III) promoter expression shRNA.Can prepare the pRS-shRNA plasmid by standard method well known in the art.In one embodiment, pRS-shRNA is from library plasmid set deconvolution, and this is by gathering transform bacteria with this, and seeks the clone who only contains interested plasmid.Preferably, 19 aggressiveness siRNA sequences are used with the suitable forward and the reverse primer that are used for sequence-specific PCR.Identify plasmid by sequence-specific PCR, and confirm by order-checking.By express the cell of shRNA with the suitable cell preparation of pRS-shRNA plasmid transfection.By suitable marker, select cell as tetracycline, and keep up to colony obvious.Selected clone then, and detect being used to and knock out.In another embodiment,, express shRNA as pRS-shRNA by plasmid.By knocking out that pRS-shRNA carries out, can be by realizing with Lipofectamine 2000 (Invitrogen) transfectional cell.
In another approach, can be delivered to animal with sending in the siRNA body, in the organ or tissue as the people (referring to, Song et al.2003 for example, Nat.Medicine 9,347-351; Sorensenet al., 2003, J.Mol.Biol.327,761-766; Lewis et al., 2002, Nat.Genetics32,107-108 introduces all above-mentioned documents as a reference in full at this).In the method, with the solution intravenous injection of siRNA in animal.SiRNA can arrive purpose organ or tissue then, and effectively reduces the expression of target gene in animal organ or tissue.
Can use any suitable propagation well known in the art or growth-inhibiting to measure the cell growth.In a kind of preferred embodiment, with the MTT proliferation assay (referring to, vande Loosdrechet for example, et al., 1994, J.Immunol.Methods 174:311-320; Ohno et al., 1991, J.Immunol.Methods 145:199-203; Ferrari et al., 1990, J.Immunol.Methods 131:165-172; Alley et al., 1988, CancerRes.48:589-601; Carmichael et al., 1987, Cancer Res.47:936-942; Gerlier et al., 1986, J.Immunol.Methods 65:55-63; Mosmann, 1983, J.Immunological Methods 65:55-63) measures the effect of one or more reagent in cell growth inhibiting.Handle the selected time period of cell with one or more candidate agents of selected concentration, as 4-72 hour.The 3-of cell and appropriate amount (4,5-dimethylthiazole-2-yl)-2 then, 5-phenylbenzene tetrazolium bromide (MTT) be the selected time period of incubation together, as 1-8 hour, makes the cell of survival that MTT is converted into insoluble first
Cell in settling.After removing the excessive MTT that comprises in the supernatant liquor, add suitable MTT solvent, as DMSO solution, with the dissolving first
By determining for example optical density(OD) at 570nm place, measure the proportional MTT concentration of number then with survivaling cell.Can measure the candidate agent of a large amount of different concns, so that determine to cause the concentration of 50% candidate agent that suppresses.
In another kind of preferred embodiment, use the alamarBlue of cell proliferation
TMMeasure screen can be used for cytostatic one or more candidate agents (referring to, Page etal. for example, 1993, Int.J.Oncol.3:473-476).AlamarBlue
TMMeasure and measure cellular respiration, and measuring used as viable cell number.The interior environment of proliferative cell has higher reductibility than the interior environment of non-proliferative cell.For example, in breeding, NADPH/NADP, FADH/FAD, the ratio of FMNH/FMN and NADH/NAF increases.These metabolic intermediates can reduce alamarBlue, therefore, can be used to monitor cell proliferation.Cell number in the sample of the processing of measuring by alamarBlue can be expressed as the per-cent with respect to the cell number in the untreated control sample.Can measure the alamarBlue reduction by absorbancy or spectrophotofluorimetry.In one embodiment, determine the alamarBlue reduction, and adopt following formula to be calculated as reduction per-cent by absorbancy:
Wherein:
λ
1=570nm
λ
2=600nm
(ε
Redλ
1)=155,677 (molar extinction coefficient of the reductive alamarBlue of 570nm place)
(ε
Redλ
2)=14,652 (molar extinction coefficient of the reductive alamarBlue of 600nm place)
(ε
Oxλ
1)=80,586 (molar extinction coefficient of the alamarBlue of 570nm place oxidation)
(ε
Oxλ
2)=117,216 (molar extinction coefficient of the alamarBlue of 600nm place oxidation)
(A λ
1The absorbancy of)=570nm place test hole
(A λ
2The absorbancy of)=600nm place test hole
(A ' λ
1)=570nm contains at the place that substratum adds alamarBlue but the absorbancy of not adding the negative control hole of cell
(A ' λ
2)=600nm contains at the place that substratum adds alamarBlue but the absorbancy of not adding the negative control hole of cell.Preferably, deduct the not % reduction in celliferous hole from the % reduction in the hole of containing sample, to determine to be higher than the % reduction of background.
Can adopt standard method well known in the art to carry out cell cycle analysis.In one embodiment, the supernatant liquor with each hole merges with the cell of gathering in the crops by tryptic digestion.Then with the proper speed centrifugal mixture.Use the 70% ice-cold ethanol time period that cell fixation is suitable then, 30 minutes according to appointment.Can wash the fixed cell once with PBS, resuspended then, for example be resuspended in 0.5ml and contain iodate third ingot (among the PBS of 10 micrograms/ml) and RNA enzyme A (1mg/ml), in suitable temperature, as 37 ℃ of time periods that following incubation is suitable, as 30 minutes.Carry out flow cytometry with flow cytometer.In one embodiment, measure necrocytosis with inferior G1 cell mass.For example, if, think that then cell is to the reagent sensitivity with the inferior G1 cell mass of the sample of agent treated inferior G1 cell mass greater than the sample of not using agent treated.
5.3.KSP the purposes of interaction gene and product thereof
The invention provides and utilize and the interactional gene of KSP (" KSP interaction gene "), identify and the albumen of KSP interaction gene or protein-interacting or the method and composition of other molecule as STK6 or TPX2 gene, its product and antibody.In a kind of preferred embodiment, the invention provides STK6 or TPX2 gene as described KSP interaction gene.The present invention also provides and has utilized the KSP interaction gene, screens the interactional method and composition of regulating the expression of KSP interaction gene or regulating KSP interaction gene or albumen and other albumen or molecule as STK6 or TPX2 gene, product and antibody.The present invention also provides and has utilized the KSP interaction gene, screens as STK6 or TPX2 gene, product and antibody can be used for regulating to the Growth Inhibition resistance of KSP inhibitor (KSPi) and/or strengthen the Growth Inhibition method and composition of KSP inhibitor cell or organism.The present invention also provides and has utilized the KSP interaction gene, as STK6 or TPX2 gene, product and antibody diagnose by the mediation of KSP interaction gene to the Growth Inhibition resistance of KSP inhibitor and be used for and use the method and composition of the therapy combination therapy disease of KSP inhibitor.
5.3.1. determine method with KSP interaction gene or the interactional albumen of its product or other molecule
Can use any method that is suitable for detecting protein-protein interaction to identify the KSP interaction protein, as the interaction between STK6 or TPX2 albumen and the another kind of cell protein.Also can adopt method well known in the art to determine the KSP interaction gene, as the interaction between STK6 or TPX2 gene and other cellular elements.
Operable traditional method comprises co-immunoprecipitation, crosslinked and by gradient or chromatography column copurification.Utilization makes it possible to identify and the interactional cell protein of KSP interaction gene product such as these program.In case obtain separating, described cell protein can be identified, can be used in combination with standard technique then, be used for identifying albumen interactional with it.For example, can use technology well known in the art, as the Edman degradation technique (referring to, Creighton for example, 1983, " Proteins:Structures and Molecular Principles ", W.H.Freeman﹠amp; Co., N.Y. pp.34-49) determines at least a portion aminoacid sequence with the interactional cell protein of KSP interaction gene product.Can be with the guidance of the aminoacid sequence that obtains as the oligonucleotide mixture that produces the gene order that can be used to screen the described cell protein of coding.For example, can finish screening by standard hybridization or round pcr.The technology that is used to prepare oligonucleotide mixture and screening be well known in the art (referring to, Ausubel for example, supra., and PCR Protocols:A Guide to Methods and Applications, 1990, Innis, M.et al., eds.Academic Press, Inc., New York).
In addition, can use the gene that causes while identification code and the interactional cell protein of KSP interaction protein.These methods comprise, for example, utilize the similar mode of antibody Detection Techniques with known λ gt11 library, with the KSO interaction protein detection expression library of mark.
Describe the interactional a kind of method in the proteic body that detects in detail, promptly two heterological systems, just in order to illustrate, rather than restriction.Described this system a kind of form (Chien etal., 1991, Proc.Natl.Acad.Sci.USA, 88:9578-9582), and can (Palo Alto CA) is purchased from Clontech.
In brief, adopt such system, made up the plasmid of the two kinds of hybrid proteins of encoding: a kind of DNA by the transcription activating protein that merges with KSP interaction gene product combines the territory and forms, another kind of by with this plasmid of recombinating as the part in cDNA library in the activation domain of the transcription activating protein that merges of the agnoprotein of cDNA coding form.DNA is transformed into the Saccharomyces cerevisiae strain that contains reporter gene (as HBS or lacZ) in conjunction with territory fusion plasmid and cDNA library, and the regulatory region of described yeast strains contains the binding site of transcriptional activator.Any independent hybrid protein all can not activate transcribing of reporter gene: DNA in conjunction with the territory hybrid protein can not because it does not provide mobilizing function, the activation domain hybrid protein can not because it can not navigate to the binding site of activator.Functional activator has been rebuild in the interaction of two kinds of hybrid proteins, and causes the expression of reporter gene, and the mensuration by the reporter gene product can detect it.
Two heterological systems or relevant method can be used for screening and the interactional proteic activation domain of " bait " gene product library.Illustrate, but be not restriction, KSP interaction gene product can be used as the bait gene product.Total genome or cDNA sequence merge with the DNA of coding activation domain.Combine this library of merging in the territory with DNA and reported in the strain to yeast by cotransfection, express those of reporter gene in the transformant that screening obtains with the plasmid of the heterozygote of coding bait KSP interaction gene product.For example, but be not restriction, bait KSP interaction gene sequence as the encoding sequence of KSP interaction gene, can be cloned in the carrier, makes its translation be blended in the DNA of the proteic DNA of coding GAL4 in conjunction with the territory.These bacterium colonies of purifying separate and are responsible for the library plasmid that reporter gene is expressed.Identify by the plasmid-encoded albumen in library with dna sequencing then.
The cDNA library that can prepare clone with the conventional method of implementing in this area, from this library with the interactional albumen of bait KSP interaction gene product be to be detected.According to particular system described herein, for example, the cDNA fragment can be inserted carrier, make their translations be blended in the transcription activating domain of GAL4.This library can with bait KSP interaction gene-GAL4 fusion plasmid cotransformation in yeast strains, this yeast strains contains the lacZ gene by the promoters driven that comprises the GAL4 activation sequence.Interactional with bait KSP interaction gene product, by the albumen that is blended in the GAL4 transcription activating domain of cDNA coding, with the active GAL4 albumen of reconstruct, and therefore drive the HIS3 expression of gene.Can detect the bacterium colony of expressing HIS3 by containing based on the growth on the Petri dish of the substratum of the shortage Histidine of semi-solid agar.Then can be from these bacterial strain purifying cDNA, and with the conventional technology production of implementing in this area with separate bait KSP interaction gene-interaction protein.
Can determine interaction between KSP interaction gene and the conditioning agent thereof by standard method well known in the art.
5.3.2. the method for screening reagent
The invention provides screening and regulate the KSP interaction protein, as the expression of STK6 or TPX2 or regulate itself and other albumen or the interactional method of molecule.
5.3.2.1. screening assay
Designed following mensuration, to identify some compounds, it is incorporated into KSP interaction gene or gene product, is incorporated into interactional other cell protein of KSP interaction gene product, is incorporated into the cellular component that influenced by KSP interaction gene product, as albumen or be incorporated into the compound of the activity (that is, regulating the activity level of STK6 or TPX2 gene expression dose and/or adjusting STK6 or TPX2 gene product) of the interactional compound that disturbs KSP interaction gene or gene product and other cell protein and adjusting KSP interaction gene.Can also utilize the mensuration of identifying the compound that is incorporated into KSP interaction gene adjusting sequence (as promoter sequence), referring to, Platt for example, K.A., 1994, J.Biol.Chem.269:28558-28562 is incorporated herein by reference in full at this, and described compound can be regulated the expression level of KSP interaction gene.Described compound can include, but not limited to influence the little organic molecule of other expression of gene that KSP interaction gene or some participations comprise the approach of KSP interaction gene, or other cell protein.The method of identifying described cell protein is described in above 5.3.1 joint.Described cell protein can participate in the Growth Inhibition of KSP inhibitor and regulate.In addition, in these compounds, comprised the expression that influences the KSP interaction gene and/or the activity of its gene product, and can be used to regulate compound the Growth Inhibition resistance of KSP inhibitor.
Described compound can include, but not limited to peptide, as soluble peptide, includes, but not limited to the member of the fusogenic peptide and the random peptide library of Ig tail; (referring to, Lam for example, K.S.et al., 1991, Nature 354:82-84; Houghten, R.et al., 1991, Nature 354:84-86), and combinatorial chemistry deutero-molecular library, described library by D-and/or L-configuration amino acid form, phospho-peptide (comprises, but be not limited at random or the phospho-peptide library part degeneracy, directed, referring to, Songyang for example, Z.etal., 1993, Cell 72:767-778), antibody (includes, but are not limited to polyclone, mono-clonal, humanization, antiidiotype, chimeric or single-chain antibody and FAb, F (ab ')
2With FAb expression library fragment and epi-position binding fragment thereof) and little organic or inorganic molecule.
Through being used for, for example, regulate the biological function of KSP interaction gene product, and be used to improve the Growth Inhibition resistance of KSP inhibitor and/or the growth-inhibiting effect of enhancing KSP inhibitor as mensuration compounds identified described herein.The 5.3.2.2. joint that is determined at hereinafter that is used for the validity of detection compound is discussed.
Can design vitro system, to identify the compound that can be incorporated into KSP interaction gene product of the present invention.Compounds identified can be used for, for example, regulate the activity of wild-type and/or KSP interaction gene product mutant, can be used to illustrate the biological function of KSP interaction gene product, can identify that destroying normal KSP interaction gene product interacts, or use in the screening of the described interactional compound of autoclasia.
The principle that is used for identifying the compound that is incorporated into KSP interaction gene product is included in the reaction mixture that is enough to make KSP interaction gene product and test compounds interaction and bonded condition and these two kinds of compositions of time preparation, forms the mixture that can remove in reaction mixture and/or detect thus.Can adopt multiple mode to carry out described mensuration.For example, a kind of method of measuring comprises KSP interaction gene product or test substances is anchored on the solid phase, and detect the KSP interaction gene product/test compounds mixture that is anchored on the solid phase when reaction finishes.In described method in one embodiment, KSP interaction gene product can be anchored on solid surface, and the direct or indirect mark test compounds of grappling not.
In practice, can easily microtiter plate be used as solid phase.Can be by non-covalent or covalent attachment, the one-tenth of grappling is separation-immobilized.Can finish non-covalent adhering to by also dry with the protein solution bag simply by solid surface.Perhaps, can be with treating the proteic specificity immobilized antibody of fixed, preferred monoclonal antibody anchors to solid surface with albumen.Can prepare surface and storage in advance.
In order to carry out this mensuration, loose composition is added the surface of the bag quilt of the composition that contains grappling.After reaction is finished, remain fixed at the alloy that forms remove under the condition of solid surface unreacted composition (as, by washing).Can adopt number of ways to finish the detection that is anchored on the mixture on the solid surface.When preliminary making during front loose composition, the detection that is fixed on lip-deep mark shows and has formed mixture.When not having the loose composition in preliminary making front, can detect with indirect labelling and be anchored on lip-deep mixture, for example, adopt the specific marker antibody (antibody can carry out direct mark or indirect labelling with the anti-Ig antibody of mark subsequently) of the loose composition in front.
Perhaps, can in liquid phase, react,, and detect mixture from unreacted component separating reaction product; For example, the alloy that forms in the specificity immobilized antibody grappling solution with KSP interaction gene product or test compounds is with the mixture of the specific marker antibody test grappling of other composition of possible mixture.
KSP interaction gene or gene product can be in vivo with one or more cells in or extracellular molecules, as protein-interacting.Described molecule can include, but not limited to nucleic acid molecule and the albumen by the method evaluation of 5.3.1 joint description as mentioned.For the purpose of discussing, described molecule is called " binding partners " at this.The activity that the bonded compound of destruction KSP interaction gene product can be used to regulate KSP interaction gene product.The bonded compound that destroys KSP interaction gene product can be used to regulate the expression of KSP interaction gene, for example passes through the combination of the conditioning agent of adjusting KSP interaction gene.Described compound can include, but are not limited to the peptide of all joints of 5.3.2.1 as mentioned description etc., and it can be near KSP interaction gene product.
Be used for identifying and disturb in KSP interaction gene product and the cell thereof or the ultimate principle of the mensuration system of the interactional compound of extracellular binding partners is included in and is enough to that KSP interaction gene product and binding partners are interacted and bonded condition and time preparation comprise the reaction mixture of these two kinds of compositions, form mixture thus.For the inhibition activity of test compounds, preparation feedback mixture under test compounds existence and non-existent condition.Test compounds can be included in the reaction mixture at first, or can add in the time after adding KSP interaction gene product and binding partners thereof.Do not having under the condition of test compounds or with placebo incubation control reaction mixture.Detect the formation of alloy between KSP interaction protein and the binding partners then.In control reaction, form mixture, but in containing the reaction mixture of test compounds, do not form mixture, show that compound disturbs the interaction between KSP interaction protein and the interactional binding partners.In addition, the mixture in the reaction mixture that contains test compounds and normal KSP interaction protein forms, and also can form with the mixture in the reaction mixture that contains test compounds and sudden change KSP interaction protein to compare.Identify to destroy mutant at needs, but do not destroy under the situation of compound of normal KSP interaction protein, this relatively is important.
Can disturb the interactional compound of KSP interaction gene product and binding partners thereof with heterogeneous or homogeneous phase form.Heterogeneous assays comprises KSP interaction gene product or binding partners is anchored on the solid phase, and detect the mixture that is anchored on the solid phase when reaction finishes.In homogeneous determination, in liquid phase, carry out entire reaction.In any method, can change the order that adds reactant, to obtain different information about the compound of test.For example, can identify by the following method by for example competing the interactional test compounds of disturbing between KSP interaction gene product and the binding partners: in the presence of test substances, react, promptly by before adding KSP interaction protein and interactional binding partners or simultaneously test substances is added reaction mixture.Perhaps,, can detect the test compounds of destroying preformed mixture, as the compound with higher binding constant of one of the composition of replacing mixture by after forming mixture, test compounds being added reaction mixture.Various forms has hereinafter briefly been described.
In the heterogeneous assays system, KSP interaction gene product or interactional binding partners are anchored on the solid phase, and the direct or indirect mark material of grappling not.In practice, can utilize microtiter plate easily.Can be by non-covalent or covalent attachment, substance fixed with grappling.Can finish non-covalent adhering to simply by also dry by solid surface with KSP interaction gene product or binding partners solution bag.Perhaps, can material be anchored to solid surface with the specificity immobilized antibody for the treatment of the fixed material.Can prepare surface and storage in advance.
In order to carry out this mensuration, be with or without the surface that under the condition of test compounds the mating partner of fixed material is exposed to the bag quilt.After reaction is finished, remove unreacted composition (for example, by washing) and remain fixed in the mixture of any formation of solid surface.Can finish the detection of the mixture that is anchored on solid surface with multiple mode.When mark in advance during loose material, the detection that is fixed on lip-deep mark shows and has formed mixture.When not in advance during the loose material of mark, can detect with indirect labelling and be anchored on lip-deep mixture, for example, adopt the specific marker antibody (antibody can carry out direct mark or indirect labelling with the anti-Ig antibody of mark subsequently) of initial loose material.According to the order of adding reacted constituent, can detect and suppress the test compounds that mixture formed or destroyed preformed mixture.
Perhaps, can in liquid phase, react,, and detect mixture from unreacted component separating reaction product existing or not existing under the condition of test compounds; For example, the alloy that forms in the specificity immobilized antibody grappling solution with one of binding constituents is with the mixture of the specific marker antibody test grappling of other mating partner.Equally, according to adding the order of reagent, can identify the test compounds that suppresses mixture formation or destroy preformed mixture to liquid phase.
In a kind of alternate embodiment of the present invention, can use homogeneous determination.In the method, the preformed mixture of preparation KSP interaction protein and interactional binding partners, wherein mark KSP interaction gene product or its binding partners, but because mixture forms, the signal that this mark produces by cancellation (referring to, the U.S. Patent No. 4,109 of Rubenstein for example, 496, it utilizes this method to carry out immunoassay).With the competition of one of material in the preformed mixture and replace the interpolation of the test substances of this material, will cause producing the signal that is higher than background.In this way, can identify the interactional test substances of destruction KSP interaction protein/binding partners.
In a kind of specific embodiment, can prepare KSP interaction gene product, fix to adopt recombinant DNA technology.For example, can adopt fusion vector, as pGEX-5X-l, make the coding region and glutathione-S-transferase (GST) gene fusion of KSP interaction gene, amalgamation mode makes and keeps it in conjunction with activity in the fusion rotein that obtains.Can the interactional binding partners of purifying, and be used to adopt the conventional method of implementing in this area to produce monoclonal antibody.For example, can use radio isotope by the conventional method of implementing in this area
125The I traget antibody.In heterogeneous assays, gst fusion protein can be anchored to gsh-sepharose 4B as GST-STK6 or GST-TPX2 fusion rotein.Then, can with allow to take place interact and the bonded mode test compounds exist or non-existent condition under the interactional binding partners of adding.When reaction finishes, can the unconjugated material of flush away, can monoclonal antibody adding system with mark in, and it is combined with the compound composition.Can keep and the associating radioactive amount of gsh-sepharose 4B by measuring, detect the interaction between KSP interaction protein and the interactional binding partners.Test compounds successfully suppresses interactional, and the radioactivity that will cause measuring reduces.
Perhaps, can under the condition that does not have solid gsh-sepharose 4B,, be mixed together in the liquid as GST-STK6 gene fusion albumen and interactional binding partners with fusion rotein.Can or add test compounds in these matter interactions afterwards.Then, this mixture can be added gsh-sepharose 4B, the unconjugated material of flush away.Equally, antibody that can be by adding mark and measurement and the associating radioactivity of pearl detect the interactional inhibition degree of KSP interaction gene product/binding partners.
In another embodiment, can adopt substituting in these two kinds of full-length proteins one or both in conjunction with the peptide fragment in territory and use these technology (is under the proteic situation at binding partners) corresponding to KSP interaction protein and/or interactional binding partners.Can use the method for the conventional any number implemented in this area to identify and the separation and combination site.These methods include, but not limited to the mutagenesis of gene of one of proteins encoded and screening coimmunoprecipitation measure in bonded destroy.Can select then to encode anaphragmic in the gene of second kind of material in the mixture.The proteic separately Gene Sequence Analysis of encoding will disclose corresponding to the proteic zone that participates in the interactional combination.Perhaps, can a kind of albumen be anchored to solid surface with method as described above in this section, and it is also combined with its binding partners interaction, described binding partners has been used proteolytic ferment, as trypsin treatment.After the washing, comprise that small peptide in conjunction with the mark in territory can keep and solid material associates, it can separate and identify by amino acid sequencing.Equally, in case obtained the gene of coding binding partners, can carry out the peptide fragment of through engineering approaches with expressing protein to short gene fragment, it is active to detect described segmental combination then, and purifying or synthetic.
For example, but not restriction, can be according to above description in this section, by preparation GST-STK6 or GST-TPX2 fusion rotein and it is combined with the glutathione agarose pearl and STK6 or TPX2 gene product are anchored to solid material.Can use radio isotope,, and use proteolytic ferment, cut as trypsinase as the interactional binding partners of 35S mark.Cleaved products can be added the GST-STK6 or the GST-TPX2 fusion rotein of grappling then, and make in conjunction with taking place.Behind the unconjugated peptide of flush away, can represent the bond material of the mark in binding partner binds territory by the known method wash-out, purifying, and analysis of amino acid sequence.The peptide that produces evaluation like this be can synthesize, or recombinant DNA technology and suitable facilitation albumen fusion adopted.
5.3.2.2. the Growth Inhibition compound of KSP inhibitor is regulated and/or is strengthened in screening
Can further screen the expression of adjusting KSP interaction gene and/or interactional any reagent of KSP interaction protein, as the adjusting and/or the Growth Inhibition abilities of enhancing KSP inhibitor in cell such as antibody of 5.3.2.1 joint compounds identified, KSP interaction protein.Any suitable propagation well known in the art or growth-inhibiting mensuration can be used for this purpose.In one embodiment, candidate agent and KSP inhibitor are applied to the cell of clone, and definite Growth Inhibition changes.Preferably, determine that with the combination of the candidate agent of different concns and different concns KSPi Growth Inhibition changes, so that determine to cause one or more combinations of the concentration of 50% candidate agent that suppresses and KSPi, that is, and IC50.
In a kind of preferred embodiment, with the MTT proliferation assay (referring to, van deLoosdrechet for example, et al., 1994, J.Immunol.Methods 174:311-320; Ohnoet al., 1991, J.Immunol.Methods 145:199-203; Ferrari et al., 1990, J.Immunol.Methods 131:165-172; Alley et al., 1988, Cancer Res.48:589-601; Carmichael et al., 1987, Cancer Res.47:936-942; Gerlieret al., 1986, J.Immunol.Methods 65:55-63; Mosmann, 1983, J.Immunological Methods 65:55-63) screens with the KSPi combination and be used for cytostatic candidate agent.Candidate agent and KSPi with selected concentration handled cell 4-72 hour.The 3-of cell and appropriate amount (4,5-dimethylthiazole-2-yl)-2 then, 5-phenylbenzene tetrazolium bromide (MTT) incubation 1-8 hour together makes the cell of survival that MTT is converted into insoluble first
Cell in settling.After removing the excessive MTT that comprises in the supernatant liquor, add suitable MTT solvent, as DMSO solution, with the dissolving first
By determining the optical density(OD) at 570nm place, measure the proportional MTT concentration of number then with survivaling cell.Can measure the candidate agent of a large amount of different concns, so that determine to cause 50% candidate agent that suppresses and the concentration of KSPi.
In another kind of preferred embodiment, use the alamarBlue of cell proliferation
TMMeasure screen with the KSPi combination be used for cytostatic candidate agent (referring to, Page etal. for example, 1993, Int.J.Oncol.3:473-476).AlamarBlue
TMMensuration is described in above 5.2 joints.In a kind of particular, carry out alamarBlue
TMMeasure, whether decide the transfection titre curve of siRNA of KSP interaction gene owing to select the KSPi of concentration to determine target, as 25nM (1S)-1-{[(2S)-4-(2, the 5-difluorophenyl)-2-phenyl-2,5-dihydro-1H-pyrroles-1-yl] carbonyl }-existence of 2-methyl propylamine and changing.With STK6 siRNA transfectional cell.After the siRNA transfection 4 hours, under the condition that has or do not exist KSPi, add the DMEM/10% foetal calf serum in 100 microlitres/hole, with plate incubation 68 hours under the condition of 37 ℃ and 5%CO2.From the hole, remove substratum, and with containing 10% (vol/vol) alamarBlue
TMReagent (Biosource International Inc., Camarillo, CA) and the DMEM/10% foetal calf serum (Invitrogen) of the 1M Hepes damping fluid tissue culture reagent (Invitrogen) of 0.001 volume replace.With plate 37 ℃ of incubations 2 hours, adopt then Softmax Pro 3.1.2 software (Molecular Devices) SpectraMaxplus read the plate device (Molecular Devices, Sunnyvale, CA) on, 570 and the 600nm wavelength readings.To there be or do not exist 25nM (1S)-1-{[(2S)-4-(2, the 5-difluorophenyl)-and 2-phenyl-2,5-dihydro-1H-pyrroles-1-yl] carbonyl }-compare with the reduction per-cent in the hole of the STK6siRNA transfection of certain titre and the hole of luciferase siRNA transfection under the condition of 2-methyl propylamine.The % reduction numerical value that when not having KSPi the hole of 0nM luciferase siRNA transfection is calculated is considered to 100%.
5.3.2.3. compounds identified
Compounds identified comprises in this screening has proved that selectivity regulates the compound that the Growth Inhibition ability of KSP inhibitor was expressed and regulated and/or strengthened to KSP interaction gene in the cell.These compounds include, but not limited to siRNA, antisense molecule, ribozyme, triple helical, antibody and peptide molecule, aptamrs and little organic or inorganic molecule.
Compounds identified also comprises the interactional compound of regulating KSP interaction protein and other albumen or molecule in this screening.In one embodiment, compounds identified is to regulate the interactional compound of KSP interaction protein and its interaction mating partner in this screening.In another embodiment, compounds identified is to regulate the interactional compound of KSP interaction gene and transcriptional regulatory agent in this screening.
5.3.3. diagnosis
Several different methods can be used for the assessment of diagnosis and prognostic because the KSP interaction gene, the cell that causes as the adjusting defective of STK6 or TPX2 is to the KSP inhibitor, as (1S)-1-{[(2S)-4-(2, the 5-difluorophenyl)-2-phenyl-2,5-dihydro-1H-pyrroles-1-yl] carbonyl }-the Growth Inhibition resistance of 2-methyl propylamine and be used to identify to have the experimenter who the growth-inhibiting effect of KSP inhibitor is had the tendency of resistance.
In one embodiment, this method comprises determines KSP interaction gene expression level in the cell, and the expression level that wherein is higher than predetermined threshold level shows that cell has the KSPi resistance.Preferably, predetermined threshold level is at least 2 times, 4 times, 8 times or 10 times of normal expression level of KSP interaction gene.In another embodiment, the invention provides the method for the KSPi resistance in the assessment cell, comprise and determine in the cell that by the abundance level of KSP interaction gene encoded protein, the abundance water-glass clear-cells that wherein is higher than predetermined threshold level has the KSPi resistance.In another embodiment, the invention provides the method for the KSPi resistance in the assessment cell, comprise and determine in the mammalian cell that by the activity level of KSP interaction gene encoded protein, the activity level that wherein is higher than predetermined threshold level shows that cell has the KSPi resistance.Preferably, abundance or active predetermined threshold level are the normal abundance of KSP interaction protein or at least 2 times, 4 times, 8 times or 10 times of activity level.
Described method is passable, for example, uses reagent such as KSP interaction gene nucleotide sequence and at the interaction gene product, comprises the antibody of its peptide fragment.Particularly, described reagent can be used for, and for example (1) detects the existence that suddenlys change in the KSP interaction gene, or detects overexpression or the expression deficiency of the mRNA of KSP interaction gene with respect to the normal expression level; (2) detect excess abundance or the abundance deficiency of KSP interaction gene product with respect to KSP interaction protein normal level.
For example, can carry out method described herein by the test kit that use comprises at least a specificity KSP interaction gene nucleic acid described herein or anti-KSP interaction protein antibody reagent, described method can be advantageously used in having illness relevant with the KSP interaction gene or unusual patient with diagnosis in for example clinical the setting.
In order to detect the sudden change in the KSP interaction gene, the cell of any tool nuclear can be used as the initial source of genomic nucleic acids.Expression or KSP interaction gene product in order to detect the KSP interaction gene can use any cell type or the tissue of having expressed the KSP interaction gene.
Detection technique based on nucleic acid is described in hereinafter 5.3.3.1 joint.The peptide detection technique is described in hereinafter 5.3.3.2 joint.
5.3.3.1.KSP the detection that interaction gene is expressed
Can utilize KSP interaction gene in many technology for detection cell or tissues, as the expression of STK6 or TPX2, for example existence of the cell levels of KSP interaction gene transcript and/or sudden change or shortage.Can will be used as the starting point of described determination techniques from the nucleic acid of any tool karyocyte, and can be according to well known to a person skilled in the art that the standard nucleic acid preparation procedure separates.For example, can use the expression level of one or more polynucleotide probes measurements KSP interaction gene of the nucleotide sequence that all comprises in the KSP interaction gene, determine the expression level of KSP interaction gene.In particularly preferred embodiment of the present invention, this method is used for the resistance of diagnosing human cancer to the treatment of employing KSPi.
The hybridization or the amplification assay that DNA can be used for biological sample relate to structure unusual of KSP interaction gene with detection, comprise point mutation, insertion, disappearance and chromosome rearrangement.Described mensuration can include, but not limited to Southern and analyze single-strand conformation polymorphism analysis (SSCP), dna microarray analysis and pcr analysis.
The described diagnostic method that is used to detect KSP interaction gene specific mutant can comprise, for example, contact and incubation comprise the nucleic acid of recombinant DNA molecules, cloned genes or its degeneracy variant, it is available from sample, as patient's sample or other suitable cell source derived from the nucleic acid reagent with one or more marks that comprise recombinant DNA molecules, cloned genes or its degeneracy variant, the conditions favouring of contact and incubation carries out specificity annealing with its complementary sequence in these reagent and KSP interaction gene.Preferably, the length of these nucleic acid reagents is 15-30 Nucleotide at least.Behind the incubation, with all unannealed nucleic acid from nucleic acid: remove the KSP interaction gene molecular hybridization body.Detect exist (if having any described molecule) of the nucleic acid carried out hybridization then.Adopt described detection scheme, the nucleic acid from purpose cell type or tissue can be fixed on the solid support of film for example, or be fixed on the frosting on the surface of microtiter plate for example or polystyrene bead.In this case, behind incubation, can easily the nucleic acid reagent of unannealed mark be removed.With well known to a person skilled in the art standard technique, finished the detection of the KSP interaction gene nucleic acid reagent of remaining, annealed, mark.Can compare with the annealing pattern of estimating from normal KSP interaction gene sequence with nucleic acid reagent annealed KSP interaction gene, so that determine whether to exist the sudden change of KSP interaction gene.
The alternative diagnostic methods that is used for detecting the KSP interaction gene specific nucleic acid molecule in patient's sample or other suitable cell source can comprise their amplification, for example pass through pcr amplification (at Mullis, K.B., 1987, U.S. Patent No. 4,683,202), then with the molecule that well known to a person skilled in the art the technology for detection amplification.Desired those compare under the situation of KSP interaction gene of normal copy if can be with the extension increasing sequence that obtains only contain during with nucleic acid amplification, so that determine whether to exist the sudden change of KSP interaction gene.
In the nucleotide sequence of described hybridization and/or the preferred KSP interaction gene of pcr analysis, comprise those that detect KSP interaction gene splice site sudden change existence.
In addition, can carry out known gene type assay technology, in the KSP interaction gene, carry the individuality of sudden change to identify.Described technology comprises, for example, uses restrictive fragment length polymerphism (RFLPs), and it comprises the sequence variations in one of the recognition site of specificity restriction enzyme of use.
In addition, described the method for the dna polymorphism of analyzing the sudden change that can be used for identifying the KSP interaction gene, described method is utilized the existence of the short polyphone reiterated DNA sequences of different numbers between the restriction enzyme sites.For example Weber (U.S. Patent No. 5,075,217 is introduced the document as a reference in full at this) has described the dna marker thing based on length polymorphism in the short polyphone of (dC-dA) n-(dG-dT) n tumor-necrosis factor glycoproteins block.The equipartition of estimating (dC-dA) n-(dG-dT) n block is 30,000-60,000bp.Closely adjacent marker shows altogether heredity of high frequency on the space, and particularly useful to identifying such as the transgenation of the sudden change in the KSP interaction gene and diagnosis disease and the illness relevant with sudden change in the KSP interaction gene.
Caskey etc. (U.S. Patent No. 5,364,759 is introduced the document as a reference in full at this) have described the DNA spectrum analysis that is used to detect short three and TTTC.This method comprises the extraction target DNA, as the KSP interaction gene, and the DNA that amplification is extracted, and the mark tumor-necrosis factor glycoproteins is to form the genotype collection of illustrative plates of individual DNA.
Also can measure the expression level of KSP interaction gene.For example, can separate and utilize the check of hybridization described above or round pcr known or suspect the cell type or the tissue of expressing K SP interaction gene, as the RNA of the cancer cell type of performance KSPi resistance.Isolated cells can be derived from cell culture or patient.The analysis of taking from the cell of culture can be the part that is used as based on the gene therapy technology of cell, or the check compound is to the steps necessary in the cell assessment of the effect of KSP interaction gene expression.Described analysis can disclose the quantitative and qualitative aspect of the expression pattern of KSP interaction gene, comprises activation or deactivation that the KSP interaction gene is expressed.
In a kind of embodiment of described detection scheme, from purpose RNA molecule synthesis cDNA molecule (as, by being cDNA with RNA molecule reverse transcription).Then the sequence in the cDNA is used as nucleic acid amplification reaction, as the template of uses such as pcr amplification reaction.Be selected from KSP interaction gene nucleic acid reagent as the reverse transcription of this method and the nucleic acid reagent of the synthetic initial reagent (as primer) in the nucleic acid amplification step.The preferred length of described nucleic acid reagent is 9-30 Nucleotide at least.In order to detect amplified production, can carry out nucleic acid amplification with radioactivity or nonradioactive labeling's Nucleotide.Perhaps, can prepare enough amplified productions, make and can pass through to use any suitable nucleic acid staining method, as, product shown by the standard ethidium bromide staining.
In addition, can " original position " carry out described KSP interaction gene and express and measure, that is, directly the tissue slice of the patient tissue that obtains from biopsy thing or excision thing (fixing and/refrigerated) be measured, thereby needn't be carried out nucleic acid purification.Can with the nucleic acid of KSP interaction gene as the probe in the described original position program and/or primer (referring to, Nuovo for example, G.J., 1992, " PCR In Situ Hybridization:Protocols And Applications ", Raven Press, NY).
Perhaps, if can obtain the suitable cell of capacity, can carry out standard Northern and analyze, to determine the mRNA expression level of KSP interaction gene.
Also can express, as the cell levels of KSP interaction transcript and/or the existence or the shortage of sudden change with the KSP interaction gene in the dna microarray technical evaluation cell or tissue.In described technology, one or more polynucleotide probes that all comprise the sequence of KSP interaction gene can be used to monitor the expression of KSP interaction gene.Therefore, the invention provides the dna microarray that comprises polynucleotide probes, described probe comprises the sequence of KSP interaction gene.
Any type of dna microarray technology can be used in combination with the present invention.In one embodiment, by cDNA fragment, be deposited on the suitable surface as the PCR product of full-length cDNA, ESTs etc. with the KSP interaction gene, preparation spot cDNA microarray (referring to, DeRisi et al. for example, 1996, Nature Genetics 14:457-460; Shalon etal., 1996, Genome Res.6:689-645; Schena et al., 1995, Proc.Natl.Acad.Sci.U.S.A.93:10539-11286; With Duggan et al., Nature GeneticsSupplement 21:10-14).In another embodiment, by photoetching technique from the teeth outwards original position synthetic contain with the high density oligonucleotide array of the sequence complementary oligonucleotide of KSP interaction gene (referring to, for example, Fodor et al., 1991, Science 251:767-773; Pease et al., 1994, Proc.Natl.Acad.Sci.U.S.A.91:5022-5026; Lockhart et al., 1996, Nature Biotechnology 14:1675; McGall et al., 1996, Proc.Natl.Acad.Sci.U.S.A.93:13555-13560; United States Patent(USP) Nos. 5,578,832; 5,556,752; 5,510,270; 5,858,659; With 6,040,138).The microarray technology of this form to detect single nucleotide polymorphism (SNPs) particularly useful (referring to, Hacia et al. for example, 1999, Nat Genet.22:164-7; Wang et al., 1998, Science280:1077-82).In another embodiment, by ink-jet technology from the teeth outwards original position synthetic contain with the high density oligonucleotide array of the sequence complementary oligonucleotide of KSP interaction gene (referring to, Blanchard for example, the open WO 98/41531 of disclosed international patent application on September 24th, 1998; Blanchard et al., 1996, Biosensors andBioelectronics 11:687-690; Blanchard, 1998, in Synthetic DNA Arraysin Genetic Engineering, Vol.20, J.K.Setlow, Ed., Plenum Press, New York at pages 111-123).In another embodiment, the dna microarray that allows to carry out the control of electronics severity can be used in combination with the polynucleotide probes of the sequence that comprises the KSP interaction gene (referring to, for example U.S. Patent No. 5,849, and 486).
5.3.3.2.KSP the detection of interaction gene product
Can be according to description herein, with diagnosis and the prognosis agent of the antibody of the KSP interaction gene product of anti-wild-type or sudden change or its conservative variant or peptide fragment as the KSPi resistance.Described diagnostic method can be used to detect expression level unusual of KSP interaction gene, or time, tissue, cell or the Subcellular Localization of the structure of KSP interaction gene product and/or product is unusual.
Because KSP interaction gene product is intracellular gene product, the illness that antibody described below and method of immunity cause in the adjusting defective of assessment treatment KSP interaction gene has importance in the external application as the effect of proliferative disease.Antibody as mentioned below or antibody fragment can be used for the possible treatment compound of in-vitro screening, to determine their effects to expression of KSP interaction gene and the production of KSP interacting peptide.Can identify the compound that the relevant illness of the interactional adjusting defective of KSP is had beneficial effect, and determine the treatment effective dose.
Also can use for example external method of immunity to assess effectiveness based on the gene therapy of cell to the relevant illness of the adjusting defective of KSP interaction gene.Antibody that can the anti-KSP interacting peptide of external use is determined the KSP interaction gene expression level finished in the cell that produces the KSP interacting peptide genetically engineered.Evidence disclosed herein shows that KSP interaction gene product is a gene product in the cell, and described mensuration is preferably carried out with cell lysate or extract.Described analysis makes it possible to determine to obtain the necessary transformant number of interior curative effect, and the optimized gene replacement scheme.
Tissue to be analyzed or cell type generally include those of known or suspection expressing K SP interaction gene, as, KSPi resistance cancer cell type.The protein separating method of Shi Yonging can be herein, for example, those that describe among Harlow and the Lane (Harlow, E.andLane, D., 1988, " Antibodies:A Laboratory Manual ", Cold SpringHarbor Laboratory Press, Cold Spring Harbor, New York), be incorporated herein by reference in full at this.Isolated cells can be derived from cell culture or patient.The analysis of taking from the cell of culture can be used to check the effect of compound to the expression of KSP interaction gene.
The preferred diagnostic method that is used to detect KSP interaction gene product or its conservative variant or peptide fragment can comprise, for example immunoassay, wherein the interaction partners by KSP interaction gene product or its conservative variant or peptide fragment and anti-KSP interaction gene product specific antibody its detect.
For example, antibody or the antibody fragment in conjunction with the KSP interaction protein can be used for the existence of KSP interaction gene product or its conservative variant or peptide fragment is carried out quantitatively or qualitative detection.This can (referring to this joint hereinafter) immunofluorescence technique be finished by for example use and opticmicroscope, flow cytometry or the fluorescently-labeled antibody of fluoroscopic examination bonded.If described KSP interaction gene product is at cell surface expression, described technology is particularly preferred.
Be used for antibody of the present invention (or its fragment) and can additionally carry out the histology use, as be used for immunofluorescence or immunoelectron microscope, be used for the in situ detection of KSP interaction gene product or its conservative variant or peptide fragment.Can be by removing histological specimen from the patient, and, finish in situ detection with its antibody that is applied to mark of the present invention.Preferably cover on the biological sample and administration of antibodies (or fragment) by antibody (or fragment) with mark.By using described program, not only can determine the existence of KSP interaction gene product or its conservative variant or peptide fragment, also can determine its distribution in being examined tissue.Employing the present invention those skilled in the art will readily recognize that, can improve in the multiple Histological method (as dyeing procedure) any one, to finish described in situ detection.
There is incubation sample down in the antibody that the immunoassay of KSP interaction gene product or its conservative variant or peptide fragment are usually included in the detectability mark that can identify KSP interaction gene product or its conservative variant or peptide fragment, as biofluid, tissue extract, the cell of new results or the lysate of the cell that incubation is crossed in cell culture, and by any technology for detection bonded antibody well known in the art.
Can make biological sample contact and be fixed on solid support or carrier, as nitrocellulose or other can fixed cell, on the solid support of cell granulations or soluble protein.Can handle with the KSP interaction protein specific antibody of detectability mark subsequently with suitable damping fluid washing upholder then.Can wash solid support to remove unconjugated antibody with damping fluid for the second time then.Can detect the amount of the bonded marker on the solid support then by ordinary method.
" solid support or carrier " is intended to represent can conjugated antigen or any upholder of antibody.Known upholder or carrier comprise glass, polystyrene, polypropylene glycol, polyoxyethylene glycol, dextran, nylon, amylase, natural and Mierocrystalline cellulose, polyacrylamide, gabbro and the magnetite modified.The character of carrier can be soluble to a certain degree or insoluble, to be used for purpose of the present invention.Support material can have any possible node configuration substantially, as long as the link coupled molecule can conjugated antigen or antibody.Therefore, the upholder configuration can be a spheric, as pearl or cylindrical, as is positioned at the outside surface of test trough internal surface or bar.Perhaps, the surface can be flat, as sheet, test strip etc.Preferred upholder comprises polystyrene bead.Those skilled in the art will know that to be used for binding antibody or antigenic many other suitable carriers, maybe can determine by using normal experiment.
Can determine the combination activity of given batch anti-KSP interaction gene product antibody according to known method.Those skilled in the art can determine each operating and optimum condition determination of measuring by normal experiment.
One of approach that can detectability mark KSP interaction gene peptide specific antibody is by it is connected with enzyme, and be used for enzyme immunoassay (EIA) (Voller, A., " TheEnzyme Linked Immunosorbent Assay (ELISA) ", 1978, DiagnosticHorizons 2:1-7, Microbiological Associates Quarterly Publication, Walkersville, MD); Voller, A.et al., 1978, J.Clin.Pathol.31:507-520; Butler, J.E., 1981, Meth.Enzymol.73:482-523; Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, FL; Ishikawa, E.etal., (eds.), and 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo).With the enzyme of antibodies will with suitable substrate, preferred chromophoric substrate reaction, reactive mode makes that generation can be by spectrophotometric for example, fluorescent method or the chemical part by the detection of visual inspection method.The enzyme that can be used to detection property traget antibody comprises, but be not limited to malate dehydrogenase (malic acid dehydrogenase), staphylococcal nuclease, Δ-5-steroidal class isomerase, Alcohol Dehydrogenase from Yeast, alpha-phosphate glyceryl ester, desaturase, triosephosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase enzymes, rnase, urease, catalase, glucose-6-phosphate dehydrogenase (G6PD), glucoamylase and acetylcholinesterase.The colorimetry of chromophoric substrate that can be by using enzyme is finished detection.Also can finish this detection by the enzymatic reaction degree of naked eyes comparison to the substrate of the standard substance of similar preparation.
Also can finish detection with in multiple other immunoassay any one.For example, by radiolabelled antibody or antibody fragment, can by use radioimmunoassay (RIA) detect the KSP interacting peptide (referring to, Weintraub for example, B., Principles ofRadioimmunoassays, Seventh Training Course on Radioligand AssayTechniques, The Endocrine Society, March 1986, are hereby incorporated by).Can be by such as using gamma counter or scintillometer or by autoradiographic method detection of radioactive isotropic substance.
Also can use the fluorescent chemicals traget antibody.When fluorescently-labeled antibody is exposed to the light time with suitable wavelength, then because fluorescence can detect its existence.In most of normally used fluorescently-labeled compounds, comprise fluorescein isothiocyanate, rhodamine, phycoerythrin, Phycocyanins, C-, allophycocyanin, Phthalyldicarboxaldehyde and fluorescamine.
Also can use the fluorescent emission metal, as
152Eu or other lanthanide series metal detectability traget antibody.Can use metal-chelating group that these metals are connected with antibody such as Diethylenetriamine valeric acid (DTPA) or ethylenediamine tetraacetic acid (EDTA) (EDTA).
Also can be by antibody and chemiluminescence compound coupling are detected antibody.Determine the existence of the antibody of chemiluminescent labeling then by the existence that detects the fluorescence that in chemical reaction process, produces.The example of useful especially chemiluminescent labeling compound is luminol,3-aminophthalic acid cyclic hydrazide, different luminol,3-aminophthalic acid cyclic hydrazide, heromatic acridinium ester, imidazoles, acridinium salt and barkite.
Equally, can use bioluminescent compound mark antibody of the present invention.Noclilucence is a class chemoluminescence of finding in the biosystem, and wherein catalytic protein has increased the efficient of chemiluminescence reaction.By detecting luminous existence, determine the existence of bioluminescent protein.The important biomolecule luminophor that is used for the mark purpose is fluorescein, luciferase and aequorin.
5.3.4. regulate the method that the KSP interaction gene is expressed
Can use and regulate the KSP interaction gene in the multiple therapy methods body, as the expression of STK6 or TPX2 according to the present invention.For example, can be to the siRNA molecular engineeringization, and be used for making in vivo KSP interaction gene silence.Also can carry out through engineering approaches, and be used for the translation of blocking-up KSP interaction RNA in the body the antisense DNA molecule.Perhaps, can design ribozyme molecule, with cutting in the body and destruction KSP interaction mRNA.In another kind of scheme, design oligonucleotides is so that hybridize with 5 ' district (district that comprises the encoding sequence upstream) of KSP interaction gene, and formation can be used to block or reduce the triple-helix structure that the KSP interaction gene is transcribed.If desired, also can design oligonucleotides so that with the binding site hybridization of negative conditioning agent with form triple-helix structure, so that the combining and strengthen transcribing of KSP interaction gene of blocking-up and negative conditioning agent.
In a kind of preferred embodiment, design siRNA, antisense molecule, ribozyme and triple helical Nucleotide are with the translation that suppresses one or more KSP interaction protein isoforms or transcribe, and may be minimum with other expression of gene influence of the total one or more motifs of KSP interaction gene to other.For reaching this purpose, should design employed oligonucleotide based on the distinctive correlated series of KSP interaction gene.
For example, but be not restriction, oligonucleotide should not drop on the highest zone of nucleotide sequence homology of nucleotide sequence and its gene of KSP interaction gene.Under the situation of antisense molecule, described sequence preference is selected from tabulation above.The length of sequence is at least 18 Nucleotide preferably, so that the enough strong annealing of realization and said target mrna sequence, thereby the translation of prevention sequence.Izant?et?al.,1984,Cell,36:1007-1015;Rosenberg?et?al.,1985,Nature,313:703-706。
Under the situation of " tup " type ribozyme, the target sequence of ribozyme is preferably selected from tabulation above.Ribozyme is the active RNA molecule of endonuclease with high special.Hammerhead ribozyme comprises at least a portion complementary hybridization region of nucleotide sequence and target RNA and is fit to the catalytic domain of cutting target RNA.Hybridization region comprises nine (9) individual or more a plurality of Nucleotide.Therefore, hammerhead ribozyme of the present invention has and sequence complementary hybridization region listed above, and length is at least 9 Nucleotide.The structure of described ribozyme and production are well known in the art, and are described in Haseloff et al., 1988, Nature, 334:585-591 more completely.
Ribozyme of the present invention also comprises RNA endonuclease (after this being called " Cech type ribozyme "), as natural existence the in Tetrahymena Thermophila (being called IVS or L-19IVS RNA), and the ribozyme (Zaug that is fully described by Thomas Cech and co-worker thereof, et al., 1984, Science, 224:574-578:Zaug and Cech, 1986, Science, 231:470-475; Zaug, et al., 1986, Nature, 324:429-433; The disclosed international patent application No.WO88/04300 of UniversityPatents Inc.; Been et al., 1986, Cell, 47:207-216).The Cech endonuclease has 8 base pair avtive spots with target RNA sequence hybridization, and the cutting of target RNA is taking place thereafter.
For with 5 ' terminal hybridization of KSP interaction gene and with its formation triple-helix structure, and can be used to block the oligonucleotide of transcribing, they preferably with impregnable other gene of expression level in 5 ' terminal sequence complementation of non-existent KSP interaction gene.Described sequence preferably do not comprise in the promotor of KSP interaction gene yet in addition slightly with the zone of described other dna homolog.Can use aforesaid compound by several different methods well known in the art, include, but not limited to use liposome as delivery vectors.When being in anti-degraded form, also can use naked DNA or RNA molecule, described anti-degraded is by modifying end, by forming ring molecule, or by using alternating bond, comprises the key that phosphorothioate bond and thiophosphoryl are modified.In addition, can pass nucleic acid, wherein nucleic acid molecule be puted together in polylysine or Transferrins,iron complexes by promoting transhipment to send.Also can pass through multiple virus vector, include, but are not limited in retrovirus, vaccinia virus, AAV and the adenovirus any one, with nucleic acid delivery in cell.
Perhaps, can make up coding or as the recombinant nucleic acid molecules of described antisense molecule, ribozyme, triple helical or KSP interaction gene nucleic acid molecule.This nucleic acid molecule can be RNA or DNA.If nucleic acid encoding RNA, this sequence preference is connected with the regulatory element operability, so that produce enough RNA products of the needs of copy.Regulatory element can allow the composing type of sequence or adjustment type to transcribe.Can be in vivo, that is, the transfer vector of one or more RNA that in the cell of biology, will encode, as bacterial plasmid or viral RNA or DNA transfection in cell.(as, Llewellyn et al., 1987, J.Mol.Biol., 195:115-123; Hanahanet al.1983, J.Mol.Biol., 166:557-580).In case be positioned at cell, transfer vector can duplicate, and is transcribed by the cell aggregation enzyme, to produce RNA, perhaps can be integrated into the genome of host cell.Perhaps, can pass through the micromanipulation technology, as micro-injection, the transfer vector transfection of sequence that will comprise one or more RNA that encode makes transfer vector or its be partially integrated in the genome of host cell in cell or transfered cell.
RNAi also can be used to knock out the expression of KSP interaction gene.In one embodiment, be used to the mRNA that degrades with the double stranded rna molecule of 21-23 Nucleotide of the homologous region of the mRNA that transcribes from KSP interaction gene hybridization, thereby make the expression " silence " of KSP interaction gene.Preferably, dsRNA has hybridization region, as, the double stranded region of 19 Nucleotide, and the sequence complementation of the encoding sequence of itself and KSP interaction gene.Any target can be decided the KSP interaction gene, be used for the present invention as the siRNA of the suitable encoding sequence of human STK6 or TPX2 gene.As exemplary embodiment, according to the Standard Selection rule (referring to, Elbashir et al. for example, 2002, Methods 26:199-213 is incorporated herein by reference in full at this) the design target decides the double-stranded siRNA of 21 Nucleotide of the coding region of KSP interaction gene.
Can use any standard method that is used to import siRNA.In one embodiment, the induced gene silence by the siRNA that decides the KSP interaction gene to the presented by cells target (referring to, for example, Elbashir et al., 2001, Nature 411,494-498; Elbashir et al., 2001, Genes Dev.15,188-200 introduces all above-mentioned documents as a reference in full at this).SiRNA can chemosynthesis, perhaps derived from by the cutting of reorganization nickase to double-stranded RNA.The another kind of method that is used to import the double-stranded DNA (dsRNA) that makes KSP interaction gene silence is shRNA, that is, short hairpin RNA (referring to, Paddison et al. for example, 2002, Genes Dev.16,948-958; Brummelkamp et al., 2002, Science296,550-553; Sui, G.et al.2002, Proc.Natl.Acad.Sci.USA 99,5515-5520 introduces all above-mentioned documents as a reference in full at this).In the method, express the siRNA that target decide the KSP interaction gene from plasmid (or virus), it is as inverted repeats, has the ring sequence that interleaves with the formation hairpin structure.The rna transcription thing that contains hair clip that obtains by nickase processing is used for reticent siRNA with generation subsequently.Can be in cell stably express based on the shRNA of plasmid, make and can in cell, carry out long-term gene silencing in vitro and in vivo (referring to, McCaffrey et al.2002, Nature 418,38-39; Xia et al., 2002, Nat.Biotech.20,1006-1010; Lewis et al., 2002, Nat.Genetics 32,107-108; Rubinson et al., 2003, Nat.Genetics 33,401-406; Tiscornia et al., 2003, Proc.AM.Acad.Sci.USA100,1844-1848 introduces all above-mentioned documents as a reference in full at this).Also target can be decided to send in the siRNA body of KSP interaction gene to be delivered to Mammals, in the organ or tissue as the mankind (referring to, Song et al.2003 for example, Nat.Medicine 9,347-351; Sorensen etal., 2003, J.Mol.Biol.327,761-766; Lewis et al., 2002, Nat.Genetics32,107-108 introduces all above-mentioned documents as a reference in full at this).In the method, with the solution intravenous injection of siRNA in Mammals.SiRNA can arrive purpose organ or tissue then, and effectively reduces target gene expression in mammalian organs or the tissue.
5.3.5. regulate the method for active and/or its approach of KSP interaction protein
The activity that can regulate the KSP interaction protein by the interaction of regulating between KSP interaction protein and its binding partners.In one embodiment, can use reagent, as the combination of binding partners as described in antibody, aptamers, the little organic or inorganic molecules in inhibiting, so that regulate the KSPi resistance.In another embodiment, can use reagent, as proteic activity in antibody, aptamers, the little organic or inorganic molecules in inhibiting KSP interaction protein adjusting approach, so that regulate the KSPi resistance.
5.3.6. decide the KSP interaction gene and/or gene product is carried out cancer therapy by target
Can be with adjusting KSP interaction gene mentioned above or albumen, suffer from the patient of cancer as STK6 or TPX2 gene or proteic expression and/or active method and/or composition and KSPi combination therapy.Particularly, described method and/or composition and KSPi can be united use, be used for the treatment of the patient who suffers from the cancer that shows KSP interaction gene or protein mediated KSPi resistance.Described therapy can be used for the treatment of cancer, includes, but not limited to rhabdosarcoma, neuroblastoma and glioblastoma, small cell lung cancer, osteosarcoma, carcinoma of the pancreas, mammary gland and prostate cancer, mouse melanoma and leukemia, and B cell lymphoma.
In preferred embodiments, method of the present invention and/or composition and KSPi unite the patient who is used for the treatment of the cancer of suffering from the KSPi resistance that shows STK6 or TPX2 mediation.In described embodiment, regulate expression and/or the activity of STK6 or TPX2, giving the susceptibility of cancer cells, thereby give or strengthen the validity of KSPi treatment to KSPi.
In combination therapy, can be before using KSPi, simultaneously or use one or more compositions of the present invention afterwards.In one embodiment, before using KSPi, use composition of the present invention.Can determine to use timed interval between composition of the present invention and the KSPi by the normal experiment that those skilled in the art are familiar with.In one embodiment, after reaching the threshold value that needs, KSP interaction protein white level gives KSPi.Can determine the level of KSP interaction protein by adopting above-described any technology.
In another embodiment, use composition of the present invention simultaneously with KSPi.
In another embodiment, also after using KSPi, use one or more compositions of the present invention.When one or more compositions of the present invention that the long half time of KSPi uses in treatment, described using can be useful especially.
It will be understood by those skilled in the art that the arbitrary combination of the different administration time that to use composition of the present invention and KSPi.For example, when the long half time of KSPi during, preferably before or after using KSPi, use composition of the present invention in composition of the present invention.
Use the frequency of composition of the present invention or the KSP interaction protein white level that needs are depended at the interval, can determine this level by above-described any technology.Change into the level that is higher or lower than needs when KSP interaction protein white level, can increase or reduce the frequency of administration of composition of the present invention.
Can by any method assessment well known in the art separately or with the effect or the benefit of the co-administered composition of the present invention of KSPi, for example, by requiring based on the dosage of measuring survival rate, side effect, KSPi or the method for its arbitrary combination.If being applied in of composition of the present invention realized any one or multiple benefit among the patient, as increase survival rate, reduce side effect, reduce the dosage requirement of KSPi, think that then composition of the present invention strengthened the KSPi treatment, and think that this method is effective.
5.3.7. treat cancer by deciding the STK6 gene with the fixed mitotic medication combined target of other target
The contriver finds that also STK6 also decides mitotic other medicines with target, interacts as taxol.Figure 18 shows STK6 makes the Hela cell to the paclitaxel treatment sensitivity.Therefore, the present invention also provides adjusting STK6 mentioned above to express and/or is active so that decide mitotic medicine with target, as taxol combination therapy cancer patients's method and composition.Particularly, described method and/or composition can be united use with taxol, suffer from the patient of the cancer of the taxol resistance that shows the STK6 mediation with treatment.Described therapy can be used for the treatment of cancer, includes, but not limited to rhabdosarcoma, neuroblastoma and glioblastoma, small cell lung cancer, osteosarcoma, carcinoma of the pancreas, mammary gland and prostate cancer, mouse melanoma and leukemia, and B cell lymphoma.
5.4. with the interactional gene of DNA disrupting agent and gene product and uses thereof
The invention provides the method and composition that in the treatment disease, utilizes with interactional gene of DNA disrupting agent and gene product.Described gene is commonly referred to as " DNA destroys response gene ".By the gene product of described genes encoding,, be commonly referred to as " DNA destroys the response gene product " as albumen.The present invention also provides expression/activity of utilizing these genes and product screening regulatory gene/gene product thereof, and/or the interactional method and composition of regulatory gene or albumen and other albumen or molecule.The present invention further provides and utilized these genes and gene product screening to be used for regulating cell the Growth Inhibition susceptibility of DNA disrupting agent and/or the Growth Inhibition compositions and methods and the composition of enhancing cell or biology DNA disrupting agent.The present invention also provides and has utilized the diagnosis of these genes and gene product to the Growth Inhibition resistance or the susceptibility of DNA disrupting agent be used for and adopt the method and composition of the therapy combination therapy disease of one or more DNA disrupting agents.
5.4.1. with interactional gene of DNA disrupting agent and gene product
The invention provides the gene that can weaken or strengthen the cell killing of DNA disrupting agent.These genes can be united use with the DNA disrupting agent of hereinafter 5.4.2 joint description.The purposes of these genes is described in hereinafter 5.4.3 and 5.4.4 joint.
In one embodiment, the invention provides the cell killing that can make the DNA disrupting agent and weaken or strengthen at least 1.5 times, 1.6 times, 1.7 times, 1.8 times and 1.9 gene, as cis, dox or campto.In a kind of preferred embodiment, the invention provides following gene, the cell killing that its silence causes the DNA disrupting agent strengthens at least 2.0 times: BRCA2, EPHB3, WEE1 and ELK1.Fig. 8 has shown that the cell killing that the silence of BRCA2, EPHB3, WEE1 and ELK1 causes the DNA disrupting agent strengthens at least 2 times.The invention provides by uniting adjusting, for example strengthen described expression of gene and/or treat method for cancer by the proteic activity of described genes encoding with comprising the therapy of using the DNA disrupting agent.
The present invention also provides the gene of the cell killing that the DNA disrupting agent that can weaken or strengthen particular type causes.Table II A shows specific gene, and its silence strengthens or weakens the wedding agent by DNA, as DNA ditch wedding agent, as the DNA minor groove binding; The DNA linking agent; Intercalator; Kill with the cell that dna adduct formation agent causes.In one embodiment, the invention provides the gene listed as Table II A, its silence makes the DNA wedding agent, cell killing as cis has strengthened at least 1.5 times, 1.6 times, 1.7 times, 1.8 times and 1.9 times, described gene such as gene I s 752-806 (1.5 times), gene I s 771-806 (1.6 times), gene I s784-806 (1.7 times), gene I s 789-806 (1.8 times) and gene I s 793-806 (1.9 times).In a kind of preferred embodiment, the invention provides following gene, its silence makes the DNA wedding agent, and the cell killing that causes as cis has strengthened at least 2 times: BRCA1, BRCA2, EPHB3, WEE1, ELK1, RPS6KA6, BRAF, GPRK6, MCM3, CDC42, KIF2C, CENPE, CDC25B and C20orf97.In another embodiment, the invention provides following gene, its silence makes the DNA wedding agent, and the cell killing that causes as cis has weakened at least 2 times: PLK (referring to Figure 16).The invention provides by uniting adjusting, for example strengthen or weaken described expression of gene and/or treat method for cancer by the proteic activity of described genes encoding with comprising the therapy of using the DNA disrupting agent.
The present invention also provides and can weaken or strengthen the topoisomerase I inhibitor, the gene of the cell killing that causes as camptothecine.In one embodiment, the invention provides the gene listed as Table II B, its silence makes the topoisomerase I inhibitor, the cell killing that causes as camptothecine has strengthened at least 1.5 times, 1.6 times, 1.7 times, 1.8 times and 1.9 times, described gene such as gene I s 635-807 (1.5 times), gene I s 673-807 (1.6 times), gene I s 702-807 (1.7 times), gene I s 727-807 (1.8 times) and gene I s 749-807 (1.9 times).In a kind of preferred embodiment, the invention provides following gene, its silence makes the topoisomerase I inhibitor, the cell killing that causes as camptothecine has strengthened at least 2 times, described gene such as NM_139286, TOP3B, WASL, STAT4, CHEK1, BCL2, NM_016263, TOP2B, TGFBR1, MAPK8, RHOK, NM_017719, TERT, ANAPC5, NM_021170, SGK2, C20orf97, CSF1R, EGR2, AATK, TCF3, CDC45L, STAT3, PRKY, BMPR1B, KIF2C, PTTG1, NM_019089, FOXO1A, STK4, SRC, ELK1, NM_018492, RASA2, GPRK6, BLK, ABL1, HSPCB, PRKACA, CCNE2, CTNNBIP1, NM_013367, FRAT1, PIK3C2A, NM_017769, XM_170783, NM_016457, XM_064050, STK6, RALBP1, ELK1, NF1, STAT5A, WEE1, PTK6, RPS6KA6, BRCA1, EPHB3 and BRCA2.In another kind of preferred embodiment, the invention provides following gene, its silence makes the topoisomerase I inhibitor, the cell killing that causes as camptothecine has strengthened at least 3 times, described gene such as XM_064050, STK6, RALBP1, ELK1, NF1, STAT5A, WEE1, PTK6, RPS6KA6, BRCA1, EPHB3 and BRCA2.In another embodiment, the invention provides following gene, its silence makes the topoisomerase I inhibitor, the cell killing that causes as camptothecine has weakened at least 2 times, described gene such as PLK, CCNA2, MADH4, NFKB1, RRM2B, TSG101, DCK, CDC5L, CDCA8, NM_006101, INSR.The invention provides by uniting adjusting, for example strengthen or weaken described expression of gene and/or treat method for cancer by the proteic activity of described genes encoding with comprising the therapy of using the topoisomerase I inhibitor.
The present invention also provides and can weaken or strengthen the topoisomerase II inhibitor, the gene of the cell killing that causes as Zorubicin.In one embodiment, the invention provides the gene listed as Table II C, its silence makes the DNA wedding agent, the cell killing that causes as Zorubicin has strengthened at least 1.5 times, 1.6 times, 1.7 times, 1.8 times and 1.9 times, described gene such as gene I s 657-830 (1.5 times), gene I s 685-830 (1.6 times), gene I s 723-830 (1.7 times), gene I s 750-830 (1.8 times) and gene I s 767-830 (1.9 times).In a kind of preferred embodiment, the invention provides following gene, its silence makes the topoisomerase II inhibitor, the cell killing that causes as Zorubicin has strengthened at least 2 times, described gene such as PTK2, KRAS2, BRA, FZD4, RASAL2, CENPE, CCNH, MAP4K3, MAP4K2, ERBB3, RHOK, MYO3A, AXIN1, INPP5D, NM_018401, NEK1, TGFBR1, XM_064050, STAT4, MAP3K1, CCNE2, STK6, HDAC4, CTNNA1, EIF4EBP1, ACVR2B, CDC42, MAPK8, BLK, WEE1, KIF26A, TCF1, NM_019089, NOTCH4, HDAC3, PIK3CB, CCNG2, TLK2, XM_066649, MCM3, ELK1, PTK6, ABL1, FZD4, XM_70783, CHUK, SRC, NM_016263 and C20orf97.In another kind of preferred embodiment, the invention provides following gene, its silence makes the topoisomerase II inhibitor, the cell killing that causes as Zorubicin has strengthened at least 3 times, described gene such as ELK1, PTK6, ABL1, FZD4, XM_170783, CHUK, SRC, NM_016263 and C20orf97.In another embodiment, the invention provides following gene, its silence makes the topoisomerase II inhibitor, and the cell killing that causes as Zorubicin has weakened at least 2 times, described gene such as PLK (referring to Figure 16).The invention provides by uniting adjusting, for example strengthen or weaken described expression of gene and/or treat method for cancer by the proteic activity of described genes encoding with comprising the therapy of using the topoisomerase II inhibitor.
In a kind of preferred embodiment, the invention provides CHEK1, BRCA1, BARD1 and RAD51 as strengthening the gene that kills and wounds of DNA disrupting agent to the p53 cell.
In another kind of preferred embodiment, the invention provides WEE1 as the gene that can weaken or strengthen the cell killing that the DNA disrupting agent causes.Wee1 is the mitotic division negative regulator of at first identifying in fission yeast-grain wine fragmentation sugar yeast (Russell and Nurse, 1987 Cell 49:559-67).The Wee1-mutant has the short G2 phase, and enters mitotic division (so called after wee) when half size of wild-type cell.In the cell of overexpression mitotic division inductor cdc25, the wee1 activity is to prevent that the lethal effect (mitotic division accident) that too early mitotic division causes is necessary.Human homology's thing of wee1 is (Igarashi et al., 1991, the Nature 353:80-83) that clones by the trans-complementation of grain wine fragmentation sugar yeast temperature dependency weel-, cdc25 overexpression mutant.The overexpression of human wee1 has produced the prolongation cell of the inhibition that changes from cell cycle G2-M in the fission yeast.This mankind wee1 clone is significantly less than its yeast counterpart, and finds to lack a part of N-terminal sequence (Watanabe et al., 1995, EMBO 14:1878-91) afterwards.
The human wee1 gene of single copy is positioned on No. 11 karyomit(e) (Taviaux and Demaille, 1993, Genomics 15:194-196).The wee1 gene is 16.96kb, has 11 exons, the mRNA transcript of coding 4.23kb.The human Wee1 albumen of 94kDa comprises 646 amino acid.According to Aceview, that is, and the integrated analysis of public obtainable experimental cDNA database (http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/av.cgi? c=locusid﹠amp; Org=9606﹠amp; 1=7465), may there be 6 kinds of littler wee1 isomer proteins that produce by alternative splicing.Multiple human cell, as lung fibroblast, embryo fibroblast, cervical cancer HeLa cell, adenocarcinoma of colon, bladder cancer (Igarashi et al., 1991, Nature 353:80-83), found the wee1 expression in uterus, blood vessel, liver, eye, spleen, bladder, skin, cartilage and the kinds of tumor cells system (UniGene, http://www.ncbi.nlm.nih.gov/UniGene/).Also found wee1 sample albumen in mouse, rat, beautiful fine rhabditida, fruit bat and Saccharomyces cerevisiae, 646 amino acid whose albumen of mouse and rat have the highest similarity degree (being respectively 89% and 91%) (UniGene).The people Wee1 sequence of total length has 5 sections with the highest PEST scoring, and the catalytic kinase domain is arranged in C-terminal (Watanabe et al., 1995, EMBO 14:1878-91).As if conservative Lys114 residue be crucial (McGowan andRussell, 1993, EMBO 12:75-85) to the weel kinase activity.
In a plurality of species, identified other Wee1 associated kinase.Africa xenopus Wee1 is that parent is expressed (ovocyte), and Wee2 expresses in zygote in non-meristem.In Mammals, relevant Myt1 has the phosphorylation activity similar to Wee1 (summarize in Kellogg, 2003, J.Cell Sci.116:4883-4890).Also identified Wee1B in the mankind, it almost only expresses (Nakanishi et al., 2000, Genes to Cells 5:839-847) in mature oocyte.
Wee1 is the nuclear Tyrosylprotein kinase of a kind of Ser/Thr of belonging to protein kinase family.Cdc-cell periodic protein B kinases when Wee1 changes by the G2/M that suppresses the cell cycle guarantees to finish dna replication dna before mitotic division.The phosphorylation of Thr14 in the ATP-binding site of Cdc2 and Tyr15 residue suppresses its activity; The Wee1 Tyrosylprotein kinase makes the Tyr15 residue phosphorylation of N-terminal.Second kind of relevant protein kinase, promptly Mik1 (Myt1) makes the Cdc2 phosphorylation on Thr14 and the Tyr15.The Cdc2 activity is that to proceed to mitotic division necessary.The dephosphorylation of crucial Tyr15 residue is catalytic by Cdc25, and Cdc25 is opposite with the effect of Wee1.The active balance of Wee1 and Cdc25 determined to enter mitotic division (summarize in Kellogg, 2003, J.Cell Sci.116:4883-4890; Pendergast, 1996, Curr.Opin.Cell Biol.8:174-181).
The Wee1 activity is regulated at the cell cycle camber.In S and G2 phase, Wee1 is active to increase parallel increase protein level.Because excessive phosphorylation and the degraded of Wee1, Wee1 activity are subjected to suppress (Watanabe et al., 1995, EMBO 14:1878-91 when mitotic division; McGowan and Russell, 1993, EMBO 12:75-85).Recently the Cdk1 (Cdc2) that studies have shown that in Africa xenopus and fission yeast can make the wee1 phosphorylation, has shown positive feedback loop model, mitotic division Cdk1 deactivation Wee1 in a small amount wherein, and caused the remarkable increase of mitotic division Cdk1 subsequently.Tome-1 also promotes reduction division to enter by decide Wee1 at G2 phase target to carry out proteolysis destruction by SCF.APC
CDH1By destroy at G1 Tome-1 and cell periodic protein B make Wee1 recover in the S phase (summarize Surana in Lim and, 2003, Mol.Cell11:845-851).
The new role of Wee1 in apoptosis proposed.The apoptotic Crk that participates in the Africa xenopus can combine with Wee1 by its SH2 structural domain.Exogenous Wee1 with Crk dependency mode quicken the Africa xenopus ovum apoptosis (Smith et al., 2000, J.CellBiol.151:1391-1400).Lacking under the nuclear output factor Crm1 bonded condition, these Crk-Wee1 mixtures also promote in the mammalian cell apoptosis (Smith et al, 2002, Mol.Cell.Biol.22:1412-1423).The research that relates to HIV albumen R (Vpr) also relate to Wee1 in the apoptosis incident (Yuan, et al., 2003, J.Virol.77:2063-2070).Vpr causes the G2 relevant with the Cdc2 deactivation to stagnate, and the G2 that prolongs stagnation causes apoptosis.Wee1 is removed in the apoptosis HeLa of Vpr inductive apoptosis HeLa cell and gammairradiation cell.The overexpression of Wee1 has weakened Vpr inductive apoptosis, and removes by the Wee1 that siRNA causes, and has induced apoptosis death.The mechanism of the obvious conflict of Wee1 level and apoptosis incident and the apoptosis-inducing that undertaken by Wee1 is not also illustrated in these researchs.
If DNA is destroyed, the effect of cell cycle inhibitor is important.Fissional blocking-up has obtained the time that DNA repairs, and make destructive DNA duplicate with separate minimum.Two cell cycles " outpost of the tax office " of genetic integrity are in G1 phase (before DNA is synthetic) and G2 phase (just before mitotic division).Lose these and close card control, promoted cell to evolve and be cancer (summarize Kastan, 1994, Science 266:1821-8) in Hartwell and.
Defective type Wee1 expresses can eliminate the G2 outpost of the tax office, promotes tumor cell proliferation.Have been found that Wee1 significantly is subjected to suppress (summarize in Lee and Yang, 2001, Cell.Mol.Life Sci.58:1907-1922) in colon cancer cell.The shortage that Wee1 expresses also relevant with relatively poor prognosis and higher nonsmall-cell lung cancer recurrence rate (Yoshida et al., 2004, Ann.Onco.15:252-256).
On the contrary, compare with sclerotic tissue on every side, Wee1 level and kinase activity be also rising (Masaki et al., 2003, Hepatology 37:534-543) in hepatocellular carcinoma.
Perhaps, the elimination at the G2 outpost of the tax office can strengthen the chemotherapy to G1 outpost of the tax office defective type tumour cell.A lot of tumour cells lack functional p53 gene, and do not show the G1 outpost of the tax office.Although normal cell will be stagnated at G1 after the DNA that radiotherapy or chemotherapy cause destroys, cancer cells will depend on the G2 outpost of the tax office and carry out DNA and repair.Therefore, the elimination at the G2 outpost of the tax office is more harmful to cancer cells comparison normal cell.Because Wee1 is to the negative adjusting of Cdc2 and weakening of Wee1 pair cell apoptosis, the chemical library screening retrieval that suppresses the compound of Wee1 with selectivity suppresses the carcinostatic agent (Wang et al., 2001, Cancer Res.61:8211-8217) at the G2 outpost of the tax office.The PD0166285Wee1 kinase inhibitor has proved the elimination of stagnating to the inhibition of Cdc2 phosphorylation, to G2 and has made the kill sensitivity of radiotherapy to the p53 mutational cell line.In one embodiment, the invention provides with PD166285 and DNA disrupting agent combination therapy method for cancer.
Wee1 activates the pathology that also may participate in rheumatoid arthritis.Rheumatoid synovial cell growth is phymatoid; Cell has a large amount of tenuigenin, maxicell nuclear and caryogram and changes.These cell transformed are present in the cartilage and bone of human rna and animal model.Rheumatoid synovial cell growth is amorphous and is anchorage independent.C-Fos/Ap-1 transcription factor in the rheumatoid synovial membrane increases.Kawasaki etc. (Kawasaki et al., 2003, Onco.22:6839-6844) proved that Wee1 is by the c-Fos/AP-1 trans-activation; Compare with the osteoarthritis cell, Wee1 significantly increases in the rheumatoid synovial cell.These synovial cells also show the tetraploid character of increase.Deactivation Wee1 can be alleviated some destruction of joint that exist among the RNA.
US20030087847A1 has described with nucleic acid molecule and has suppressed the active method of Chk1, makes the approach of p53 defective type tumour to the chemotherapy sensitivity as eliminating the G2 outpost of the tax office and selectivity.Chk1 makes the inhibition residue phosphorylation on the Cdc25, and Cdc25 is the activator of Cdc2.EP1360281A2 has described Wee1 Nucleotide and aminoacid sequence, the method that the active compound of Wee1 is regulated in the method for express recombinant Wee1 and evaluation.
In another kind of preferred embodiment, the invention provides EPHB3 as the gene that can weaken or strengthen the cell killing that the DNA disrupting agent causes.Receptor tyrosine kinase (RTK) is to have the transmembrane protein of extracellular ligand in conjunction with kinase domain in territory and the cell.Eph acceptor with 14 members comprises maximum RTK subfamily.The outside branch of Eph acceptor comprises immunoglobulin (Ig) (Ig) district (ligand binding domain) of inferring, be to be rich in the zone of halfcystine and near two repetitions of III type fibronectins (Connor and Pasquale, 1995Oncogene 11:2429-2438 the single transmembrane segment subsequently; Labrador etal., 1997, EMBO 16:3889-3897).Tenuigenin partly comprises the tyrosine kinase domain of high conservative, and its flank is lower membrane-proximal region of conservative property and C-terminal tail (sterile α motif and PDZ binding motif).According to the sequence homology of extracellular domain, the Eph acceptor is divided into two groups.The EphA acceptor is with high-affinity and ephrin-A ligand interaction, and this receptor is fixed in cell surface by glycosyl-phosphatidyl inositol (GPI) anchor.The EphB acceptor is preferential in conjunction with striding film ephrin-B part.For each group, acceptor can combine with more than one part, and each part can with more than one receptors bind.Between A and B subgroup, have less receptor-ligand cross-talk and (summarize Klein, 1997Trends in Genetics13:354-359 in Orioli and; Pasquale, 1997Curr.Biol.9:608-615).The Eph acceptor only can activate by the ephrin of film combination or artificial cluster; And the certain bind receptor of soluble ligand, they do not cause acceptor autophosphorylation (Davis et al., 1994Science 266:816-819).Eph acceptor and ephrin are unique, because the two-way signal transmission of they mediations.Because their film bonding state, Eph acceptor and ephrin are considered to mediated cell-cell interaction rather than long scope function.
The Eph receptor expression is uniqueness but eclipsed, shows uniqueness but redundant function.The Eph receptor expression is the highest in nervous tissue, but also is present in many tissues.Be expressed among the embryo of growth higherly, but also be present in the adult tissue.Receptor-ligand binding causes cellular rejection usually, and these repulsive interactions participate in developmental axon guidance, cynapse formation, neural range mode, blood vessel generation and cell migration.The neurocyte that these acceptors also can participate in growing up, blood vessel take place and tumour takes place (to summarize the Pasquale in Dodelet and, 2000Oncogene 19:5614-5619; Zhou, 1998Pharmacol.Ther.77:151-181; Pasquale, 1997Curr.Opin.Cell Biol.9:608-615).Cellular rejection or go to adhere to seemingly and transmit molecule by Eph acceptor and many signals, as the mediation of the interaction between Nck, Ras-GAP, Src, SHEP1 and the SHP2 (Wilkinson, 2001Neurosci.Rev.2:155-164).
There are 8 kinds of EphA acceptors (EphA 1-8) and 6 kinds of EphB (EphB 1-6) acceptor, their about 1000 amino acid whose albumen of all encoding.At many species, as having identified the Eph gene among chicken, rat, mouse and the mankind.EphB3 is also referred to as Hek2, Sek4, Mdk5, Cek10 or Tyro 6, it can with part ephrin-B 1-3 interact (Pasquale, 1997, Curr.Opin.Cell Biol.9:608-615).The EphB3 sequence is high conservative (>95% amino acid identity) in different plant species.The 20.2kb EphB3 gene of single copy is positioned on human No. 3 karyomit(e)s, has 16 exons.Human protein is formed (ref.seq.NM004443) by 998 amino acid.In embryo development procedure and in adult brain, enteron aisle, placenta, muscle, heart and lung and the kidney (intensity is lower in lung and the kidney), there is high-level mouse EphB3 transcript (Ciossek et al., 1995Oncogene 11:2085-2095).In Adult Human Brain, lung, pancreas, liver, placenta, kidney, skeletal muscle and heart, there is EphB3 transcript (Bohme et al, 1993Oncogene 8:2857-2862).
Identified the EphB3 splice variant in chicken, it has 15 aminoacid insertion (Sajjadi and Pasquale, 1993Oncogene 8:1807-1813) in nearly membrane structure territory.Except main 4.8kb total length EphB3 transcript, littler 2.8kb, 2.3kb and 1.9kb transcript (Ciossek et al., 1995Oncogene 11:2085-2095) in mouse tissue, have been found.In human EphB3, only observed a kind of transcript size (Bohme etal., Oncogene 1993 8:2857-2862).But, identified human EphB2 splice variant, show other isoform (Tang et al., 1998Oncogene 17:521-526) that can find other human Eph acceptor.
Having carried out considerable Eph acceptor in fetal development characterizes.(Genes ﹠amp such as Adams; Dev.13:295-306) proved that EphB3 expresses in yolk sac, and grown the artery and the vein of mice embryonic.They have proved that also the two mutant mices of EphB2/EphB3 show the defective of vitelline vesselization, the expansion of pericardium capsule, vascular development defective and head, heart and body segment blood vessel generation defective.Adams etc. have determined that also the ephrin-B part can induce the capillary vessel in the external test to sprout.
The EphB3 deficient mice has illustrated that acceptor participates in cerebral commissure, particularly connects two Interhemispheric callosal formation.In addition, the EphB2/EphB3 double-mutant has jaw and splits, and shows that they also participate in facial grow (Orioli et al., 1996EMBO 15:6035-6049).
In enteric epithelium, along with differentiation, stem cell produces the precursor with the AD HOC migration.In the intestinal epithelial cells beta-catenin white/sudden change of TCF activates and causes polyp to form.People such as Batle proved beta-catenin white/in the tcf signal transmission incident control colorectal cancer cell and express along the EphB3 of crypts-fine hair axle.In the mouse of no EphB3, migration shows the defective among the sorting cells group to occupy Paneth cell random position in crypts of intestinal crypts bottom usually.In addition, in the EphB2/EphB3 double-mutant, in intestinal epithelial cells, be mixed with the cell (Batle et al., 2002Cell 111:251-263) of propagation and differentiation.
Also in the adult mice cochlea, found the EphB3 expression, shown that it may work in peripheral auditory system.Compare with wild-type contrast, the EphB3 knock-out mice show significantly lower distortion-product ear hearing emission DPOAE level (Howard et al., 2003Hear.Res.178:118-130).The DPOAE observed value has reflected the cochlear function on the external hair cell level.
Willson etc. have proved the rise (Willson et al., 2003, Cell Transpl.12:279-290) that EphB3 expresses in the impaired spinal cord of adult rat damage location.EphB3 receptor expression co is in the CNS zone that also has high-level ephrin B part.The additional expression of damage location EphB3 acceptor and part may be because the damage back suppresses the environment of axon regeneration.
In the tumor cell line of mammary gland and epithelial origin, detected EphB3 (Bohme etal., 1993, Oncogene 8:2857-2862).Also in the kinds of tumors type, raised other Eph receptor expression level (summarizing Pasquale, Oncogene 200019:5614-5619) in Dodelet and.Some evidence promptings, the rise of Eph as if do not drive propagation (Lhotakand Pawson, 1993, Mol.Cell.Biol.13:7071-7079), as if but the expression that raises is relevant (Andres et al., 1994Oncogene 1461-1467 with metastatic potential; Vogtet al., 1998Clin.Cancer Res.4:791-797).
Organizing unordered the adhesion with abnormal cells is the sign of late tumor.It is extremely sensitive that the overexpression of Eph acceptor may make tumour that ephrin is activated, and promotes cell adhesion minimizing, cell movability and invasive.Have been found that the Eph acceptor influences cell-matrix by the adjusting integrin activity and adheres to.Maio et al. (2000Nature Cell.Biol.2:62-69) has proved on the prostate cancer cell ephrin A1 part to the activation of EphA2, the cell attachment of instantaneous inhibition of integrins mediation.In addition, among the Africa xenopus embryo, the ectopic expression of ephrin-Bl or activated EphA4 disturb the cadherin dependent cell to adhere to (Jones et al, 1998Proc.Natl.Acad.Sci.USA 95:576-581 in early days; Winning et al, 1996Dev.Biol., 179:309-319).
Set up the contact between Eph acceptor and the cell skeleton change, this is the ambulant critical aspects of cell.Activate EphB4 by the ephrin-B2 part, induced the film wrinkling (Marston et al., 2003Nat.Cell Biol.5:879-888) of Rac mediation in the Eph express cell.People such as Wahl (2000J.Cell Biol.149:263-270) have proved that ephrin-A5 induces subsiding of nerve growth circular cone in Rho dependency mode.Rho and Rac all with participate in the cell change relevant (summarizing al., 2000Exp.Cell Res.261:1-12) that tumour forms in Schmitz et.The activation of these signal pipelines that the Eph acceptor causes can cause tumour to invade and shift.
Because the effect in embryonic blood vessel growth and blood vessel generation of Eph acceptor and their part (is summarized the Bicknell in Sullivan and, 2003Br.J.Cancer 89:228-231), these molecules also can participate in tumor growth by the vascularization that causes tumour.Verified Eph receptors ligand promotes endothelial cell tissue and is assembled into capillary structure, and induces from the blood vessel that exists and carry out capillary vessel sprout (Daniel et al., 1996Kidney Intl.Suppl.57:S73-81; Pandey et al., 1995Science 268:567-569).But excretory ephrin part also can be used as the chemoattractant of the disperse of endotheliocyte; The eph acceptor of expressing on the tumour cell can instruct from the endotheliocyte that enters and make up neovascularity (Pandey et al., 1995Science 268:567-569).
Since the rise (Bohme et al., 1993Oncogene 8:2857) in tumour cell, and may participate in tumor vessel generation and transfer, and EphB3 can produce the attractive target of cancer diagnosis or treatment interference.Soluble E phA-Fc acceptor suppress the skin window measure in and the tumor vessel that suppresses in the body to have injected in the mouse of 4T1 tumour cell (Brantley et al, 2002 Oncogene 21:7011-7026) take place.
Perhaps, may there be such situation, wherein may needs to strengthen the blood vessel occurrence features of Eph acceptor, for example be used for the treatment of coronary vasodilator and block.
The expression of EphB3 also can be used as the attractive treatment target of CNS damage in the impaired spinal cord.The cellular rejection effect of EphB3 can cause impaired spinal cord aixs cylinder not regenerate.Studies have shown that axon regeneration (Bregman et al., 1995Nature 378:498-501 take place in the impaired spinal cord other minute period of the day from 11 p.m. to 1 a.m when suppress axon regeneration with antibody blocking; GrandPre et al., 2002Nature 417:547-551).
Eph acceptor autophosphorylation be subsequently with have the critical event that signal that Tyrosine O-phosphate combines the SH2 in territory transmits interaction of molecules and (summarize al, 1998Curr.Opion.Neuro.8:375-382) in Bruckner et.
People such as Binns (Binns, et al., 2000, Mol.Cell.Biol.20:4791-4805) describe the ephrin-stimulated cells that is used to study EphB2 on the neuronal cell and measured system.In brief, set up the NG108-15 clone (NG-EphB2WT cell) of stably express EphB2.The feature of NG108-15 cell performance motor neuron, motor neuron are a kind of cell types of expressing EphB2 during fetal development.But, NG108-15 cell not endogenous expression EphB2 or react on the ephrin-B part.Stimulate the NG-EphB2WT cell with Fc-ephrin-B 1, cause spinous process to disappear and the depolymerization of polymerization Actin muscle structure.Expressing cell that tyrosine to phenylalanine replaces (crucial phosphorylation site) in wild-type NG108-15 cell and the nearly film motif does not show the cell bone that reacts on ligand stimulation and reinvents.Also with anti--p Tyr antibody detection wt EphB2 and EphB2 (phosphorylation of tyrosine residues makes a variation in the transformant of Y → F).The EphB2 function of receptors weakens a kind of composition p62 that also causes eph signal transmission cascade
DokPhosphorylation reduce.
Patent US6169167 has also described with cell-cell autophosphorylation and has measured to determine the hek4 part hek4 activated method.After receptor-ligand binding, from the lysate immunoprecipitation Hek4 acceptor of the Chinese hamster ovary celI of expressing Hek4DNA.Lysate is used to the Western trace that adopts anti-phosphotyrosine antibody to carry out.
In another kind of preferred embodiment, the invention provides RAD51 as the gene that can weaken or strengthen the cell killing of DNA disrupting agent.In mammalian cell, can repair double-stranded DNA fracture (DSBs) by non-homogeneous terminal connection (NHEJ) or by homologous recombination.NHEJ relates to is not having under the situation of template the DNA end to fracture connect again, and can cause the sudden change or the disappearance of broken site.Homologous recombination needs template, complete sister's duplex, and causes the high-fidelity reparation.Homologous recombination is paused in also can DNA plerosis or the replication fork of fracture.The reparation of DSBs is important, because if they are not handled or inaccurate reparation, the impaired or apoptosis of function may take place.Genetic instability is the key feature of tumour cell, and it also can cause not having Hi-Fi homologous recombination reparation.The exchange of the initial step-autosyndetic pairing of homologous recombination and chain, relate to the albumen that belongs to RecA/Rad51 recombinase family (summarize West in Baumann and, 1998, Trends Biochem.Sci.23:247-251; Henning and Sturzbecher, 2003, Toxicology 193:91-109).
E. coli protein RecA is as the conditioning agent to DNA destructive SOS reaction, and promote autosyndetic pairing and chain exchange (summarize West in Baumann and, 1998, TrendsBiochem.Sci.23:247-251).Identified that in Saccharomyces cerevisiae DSB repairs gene rad51, and with recA homology (Shinohara et al., 1992, Cell 69:457-470).Also from human and cloned mouse rad51 gene (Yoshimura et al., 1993, NucleicAcids Res.21:1665; Shinohara et al., 1993, Nature Genet.4:239-243).The human radS1 gene of single copy is positioned at No. 15 karyomit(e)s (Shinohara et al, 1993, Nature Genet.4:239-243).The rad51 gene is made up of 10 exons, 339 the amino acid whose albumen of encoding.Two kinds of proteic aminoacid sequences of Mammals Rad51 and yeast Rad1 have 83% homology, have 56% homology with intestinal bacteria RecA albumen.Homologous region between RecA and the Rad51 comprises and is used to recombinate, UV resistance and oligomer form functional domain (the 321-260 position of RecA) (Yoshimura et al., 1993, Nucleic Acids Res.21:1665; Shinohara et al., 1993, Nature Genet.4:239-243).Finding that mouse Rad51 transcript is high level in thymus gland, spleen, testis and ovary, is low-level (Shinohara et al, 1993, Nature Genet.4:239-243) in brain.Rad51 expresses Cycle Regulation seemingly, transcribes rise (Flygare etal., 1996, Biochim.Biophys.Acta 1312:231-236) during S and G2.In addition, (Rad51B-D), they and Rad51 have 20-30% identity for XRCC2, XRCC3 to have identified 5 kinds of Rad51 parallelism homologues.These parallelism homologues can promote the Rad51 kitchen range form (summarize Schild in Thompson and, 2001, Mutat.Res.477:131-153).
Rad51 works as long spiropolymer, and it twines DNA, to form nucleoprotein filament.Rad51 is incorporated into by the molten nuclearity in DSB site and cuts the single stranded DNA that produces again, and this interaction is strengthened by Rad52.Again Qie Ge DSB end occurs in the Rad51 nucleoprotein filament to the intrusion of homoduplex, need ATP in conjunction with but do not need hydrolysis.Second end that cuts again also caught by Rad51.The end of the cutting again of invading is as DNA synthetic primer again.Holliday-engage untie be connected make the duplex of repairing can separate (summarize in West, 2003, Nat.Rev.Mol.Cell.Biol.4:435-445).People such as Pellegrini (2002, Nature 420:287-293) have reported the motif among the conservative tumor-necrosis factor glycoproteins BRC4 simulation Rad51 among the BRCA2, and as the interface that is used for the monomeric oligomerization of Rad51.Although this BRC4-Rad51 mixture is arranged, BRCA2 can control the assembling of Rad51 nucleoprotein filament.The Rad51 activity also is subjected to the adjusting of other mechanism.Find that the p53 downward modulation is by the promoted homologous recombination of Rad51 (Linke et al., 2003, Cancer Res.63:2596-2605; Yoon et al., 2004, J.Mol.Biol.336:639-654).Find that the Rad51 nucleoprotein filament that Rad54 makes double-stranded DNA (dsDNA) go up formation decomposes, and can participate in the renewal of Rad51-dsDNA silk, this is important in DNA chain permutoid reaction.In yeast, find that Srs2 suppresses reorganization (Veaute et al., 2003, Nature 423:309-312 by the Rad51 silk formation that destroys on the single stranded DNA; Krejci et al., 2003, Nature423:305-309).
Identified the splice variant of Rad51.A kind of transcript (NM_133487) lacks the interior segments corresponding to 5 ' part of exon 4,5 and exon 6, obtains lacking the albumen of 97 amino acid whose interior regions.Transcript by Genbank numbering AY425955 identification also shows the splice variant that has further brachymemma in testis.Also at other species, as found in the caenorhabditis elegant Rad51 splice variant (Rinaldo et al., 1998, Mol.Gen.Genet.260:289-294).
Some studies have shown that Rad51 135C polymorphism significantly raise risk (Levy-Lahad et al., 2001, the Proc.Natl.Acad.Sci.USA 98:3232-3236 of mammary cancer among the carrier of BRCA2 (but not being BRCA1); Kadouri et al., 2004, Br.J.Cancer 90:2002-2005).In suffering from two patients of BILATERAL BREAST CANCER, reported missense mutation (Gln150Arg), but opposite, in most of tumours, do not find Rad51 sudden change (Katoet al., 2000, J.Hum.Genet.45:133-137; Schmutte et al., 1999, CancerRes.59:4564-4569).The Rad51 knock-out mice is dead in early days in fetal development, but heterozygote survival and can educating, and can not set up rad51
-/-Mouse cell shows the keying action (Tsuzuki et al., 1996, Proc.Natl.Acad.Sci.USA 93:6236-6240) of this gene.(1998, EMBO J. is 17:598-608) by using the promotor control Rad51 transgenosis that can check to prepare rad51 for people such as Sonoda
-/-Chicken B lymphocyte DT40 clone.The genetically modified inhibition of rad51 has caused high-caliber rhexis, the cell cycle arrest of G2/M phase and necrocytosis in the DT40 cell.The overexpression of Rad51 in clone also investigated in some researchs.Vispe etc. (1998, Nucleic Acids Res.26:2859-2864) find that the Rad51 overexpression in the Chinese hamster ovary celI has increased the homologous recombination between the homology allelotrope of two adjacency, and have increased late S/G
2Cell cycle phase is to the resistance of ionizing rays.Work that people such as Richardson (2004, Oncogene 23:546-553) carry out proposed that Rad51 level in the tumour cell increases and the chromosome instability relevant with tumour progression between get in touch.The Rad51 level DSB between inductive phase in mouse ES cells system instantaneous rise 2-4 doubly produced new recombinational repair product and produced abnormal karyotype.
Reported that in tumour the Rad51 level raises, shown that the Rad51 rise can promote tumour progression.People such as Maacke (2000, Int.J.Cancer 88:907-913) have reported the positive correlation between Rad51 overexpression and breast tumor are by stages.Also having observed with non-pernicious control cells in kinds of tumor cells system is that the 2-7 that compares Rad51 doubly increases (Raderschall etal., 2002, Cancer Res.62:219-225).Also in 66% human pancreas adenocarcinoma tissue sample, found the Rad51 overexpression (Maacke et al., 2000, Oncogene19:2791-2795).Rad51 overexpression in the supposition cancer cells can be protected cell to avoid DNA destruction or cause genomic instability and diversity.The expression of also having observed Rad51 in human fibroblast's immortalization raises and recombinates to be increased (Xia et al., 1997, Mol.CellBiol.17:7151-7158).
The function of Rad51 in the tumour resistance pointed out in many researchs.People such as Hansen (2003, Int.J.Cancer 105:472-479) have proved the Etoposide resistance positive correlation in RadS1 level and small cell lung cancer (SCLC) cell.In addition, with the downward modulation of justice or antisense constructs being arranged or raising Rad51, changed the Etoposide susceptibility in the SCLC cell.The treatment of discovery Chlorambucil induces the Rad51 in the B cell chronic lymphocytic leukemia cell to express (Christodoulopoulos et al., 1999, Clin.Cancer Res.5:2178-2184).The antisense Rad51 oligonucleotide DNA that enhanced rad causes in mice embryonic skin cells system and pernicious glioma destroys (Taki et al., 1996, Biochem.Biophys.Res.Commun.223:434-438; Ohnishi et al., 1998, Biochem.Biophys.Res.Commun.245:319-324).The Rad51 downward modulation of carrying out with ribozyme has also increased prostate cancer cell to radiating susceptibility (Collis et al., 2001, Nucleic Acids Res.29:1534-1538).Interaction by the BRC tumor-necrosis factor glycoproteins on Rad51 and the BRCA2 destroys its function, also causes in the cancer cells hypersensitivity (Chen et al., 1999, J.Biol.Chem.274:32931-32935 to radiation and methyl mesylate; Chen et al., 1998, Proc.Natl.Acad.Sci.USA95:5287-5292).People such as Slupianek (2001, Mol.Ceu 8:795-806) have proved that it is important that Rad51 expresses cis-platinum in the myelocyte and ametycin resistance.These research promptings Rad51 improves the attractive target that cancer therapy is renderd a service.
5.4.2.DNA disrupting agent
Can implement the present invention with any known DNA disrupting agent, include, but are not limited to the combination of any topoisomerase enzyme inhibitor, DNA wedding agent, antimetabolite, ionizing rays or two or more described known dna disrupting agents.
The topoisomerase enzyme inhibitor that can unite use with the present invention can be topoisomerase I (TopoI) inhibitor, topoisomerase II (TopoII) inhibitor or dual topoisomerase I and II inhibitor.Topo I inhibitor can be from following any compounds: camptothecin analogues is (as karenitecin, amino camptothecin, lurtotecan, the holder pool is for bearing, irinotecan, BAY 56-3722, rubitecan, GI14721, exatecan mesylate), the rebeccamycin analogue, PNU 166148, rebeccamycin, TAS-103, camptothecine is (as the polyglutamic acid camptothecine, camptothecin sodium), intoplicine, ET 743, J-107088, pibenzimol.The example of preferred topo I inhibitor includes, but not limited to camptothecine, the holder pool replaces and bears (hycaptamine), irinotecan (irinotecan hydrochloride), belotecan, or its analogue or derivative.
Can unite the topoisomerase II inhibitor of use with the present invention can be from following any compounds: anthracycline antibiotic is (as carminomycin, Perarubicin, liposome citric acid daunorubicin, daunomycin, 4-iodo-4-deoxidation Zorubicin.Zorubicin, n, n-phenylbenzene daunomycin, the morpholino Zorubicin, the aclacinomycin microbiotic, duborimycin, menogaril, U-15167, zorubicin, pidorubicin, marcellomycin, detorubicin, annamycin, 7-cyanoquinocarcinol, the deoxidation Zorubicin, darubicin, GPX-100, MEN-10755, valrubicin, KRN5500), epipodophyllotoxin compound is (as resin of podophyllium, teniposide, Etoposide, GL331,2-ethyl hydrazides), the anthrone compound is (as the dihydro-amine anthrone, Bisantrene, mitoxantrone, anthrone), Ciprofloxacin, the carboxamide acridine, amonafide, anthracene pyrazoles microbiotic is (as teloxantrone, sedoxantronetrihydrochloride, piroxantrone, the anthracene pyrazoles, losoxantrone), TAS-103, fostriecin, tetrahydroform, XK469R, XK469, chloroquinoxaline sulfonamide, merbarone, intoplicine, elsamitrucin, CI-921, the pyrazolo acridine, hydroxyl serge carbazole, amsacrine.Preferred topoisomerase II inhibitor comprises, but is not limited to Zorubicin (adriamycin), phosphoric acid Etoposide (etopofos), teniposide, sobuzoxane, or its analogue or derivative.
Can include but not limited to DNA ditch wedding agent with the DNA wedding agent that the present invention unites use, as the DNA minor groove binding; The DNA linking agent; Intercalator; Form agent with dna adduct.The DNA minor groove binding can be an anthracycline antibiotic, the mitomycin microbiotic is (as porfiromycin, KW-2149, Mitomycin B, Mitomycin A, ametycin), chromomycin A3, carzelesin, the actinomycin microbiotic is (as cactinomycin, gengshengmeisu, actinomycin F1), brostallicin, Quinomycin A, bizelesin, duocarmycin microbiotic (as KW 2189), A Duolaixin, the Olivomycine microbiotic, plicamycin, neocarzinostatin, distamycin, MS-247, ET 743, amsacrine, anthramycin and pibenzimol, or its analogue or derivative.
The DNA linking agent includes but not limited to anti-tumor alkylating agent, 8-methoxypsoralen, mitomycin microbiotic, psoralene.Anti-tumor alkylating agent can be that the nitrourea compound is (as cystemustine, tauromustine, Semustine, PCNU, U-9889, SarCNU, CGP-6809, carmustine, Fotemustine, the methyl nitrourea, nimustine, MCNU, the ethyl nitrourea, chlorethyl cyclohexyl nitrosourea, chlorozotocin), mustargen reagent is (as mustard compound, as Spiromustine, Z-4828, Chlorambucil, Emcyt, 2,2, the 2-RA3, prednimustine, Novoembichin, Phenamet, glufosfamide, PTC, ifosfamide, Defosfamide, mustargen, phenesterin, mannomustin, endoxan, melphalan, perfosfamide, Nitromin hydrochloride, uracil mustard, bestrabucil, the DHEA mustargen, tallimustine, mafosfamide, Anilin Mustard, Chlornaphazine; The sulphur mustard compound is as the yperite thing; The mustargen prodrug, as TLK286 and ZD2767), ethylenimine compound is (as the mitomycin microbiotic, ethyleneimine, uredepa, plug is for group, diaziquone, hexamethylene bisacetamide, pentamethylmelamine, hexamethyl melamine, cardinophyllin, triaziquone, tetramethylurethimine, dualar, carboquone), sulfonic acid alkane ester cpds is (as the dimethyl busulfan, Yoshi-864, Improsulfan, piposulfan, treosulfan, busulfan, hepsulfam), epoxide compound is (as anaxirone, mitolactol, dianhydrogalactitol, Teroxirone), the miscellaneous alkylating agent is (as ipomeanol, carzelesin, methylsulfonic acid methylene ester, mitobronitol, bizelesin, U 73975, piperazinedione, VNP40101M, asaley, 6-hydroxymethyl alkyl fulvenes, E09, triethylene glycol diglycidyl ether, ET 743, pipobroman), platinic compound is (as ZD0473, liposome-cis-platinum analogue, satraplatin, BBR 3464, spiroplatin, ormaplatin, cis-platinum, RP-54780, carboplatin, lobaplatin, the folding neratein, iproplatin), triene compound is (as the imidazoles mustargen, CB 10-277, mitozolomide, temozolomide, procarbazine, Dacarbazine), picoline compound (as penclomedine), or its analogue or derivative.The example of preferred alkylating agent includes but not limited to cis-platinum, mitolactol, Fotemustine, ifosfamide, MCNU, nedaplatin (latoplatin), bendamustine (bendamustinehydrochloride), eptaplatin, temozolomide (methazolastone), carboplatin, hexamethyl melamine, prednimustine, RP-54780, carmustine, plug for group, leusulfon (busulfan), lobaplatin, endoxan, bisulfan, alkeran and Chlorambucil, or its analogue or derivative.
Intercalator can be anthrone compound, bleomycin microbiotic, rebeccamycin analogue, acridine, carboxamide acridine, amonafide, rebeccamycin, anthracene pyrazoles microbiotic, Quinomycin A, psoralene, LU 79553, BW A773U, crisnatol mesylate, benzopyrene-7,8-glycol-9,10-epoxide, acodazole, hydroxyl serge carbazole, pixantrone, or its analogue or derivative.
Dna adduct forms agent and includes but not limited to enediyne antitumor antibiotics (as dynemicin A, esperamicin A1, neocarzinostatin, dynemicin, calicheamicingamma II), platinic compound, carmustine, tamoxifen (as 4-hydroxyl-tamoxifen), psoralene, piperazine diaza oxyhydroxide, benzopyrene-7,8-glycol-9,10-epoxide, or its analogue or derivative.
Antimetabolite includes but not limited to cytosine(Cyt), cytosine arabinoside, floxuridine, Fluracil, purinethol, gemcitabine and methotrexate (MTX).
Ionizing rays includes but not limited to X ray, gamma-rays and electron beam.
5.4.3. determine to destroy the method for the interactional albumen of response gene or other molecule with DNA
Can be with being suitable for detecting any method identification of dna destruction reactive protein of protein-protein interaction and the interaction between the another kind of cell protein.Also can be with the interaction between method identification of dna destruction response gene well known in the art and other cellular elements, as the interaction between reaction of DNA destruction and the conditioning agent thereof.
Operable traditional method comprises co-immunoprecipitation, crosslinked and by gradient or chromatography column copurification.Utilization makes it possible to identify with DNA and destroys the interactional cell protein of response gene product such as these program.In case obtain separating, described cell protein can be identified, can be used in combination with standard technique then, be used for identifying albumen interactional with it.For example, can use technology well known in the art, as the Edman degradation technique (referring to, Creighton for example, 1983, " Proteins:Structures and Molecular Principles ", W.H.Freeman﹠amp; Co., N.Y., pp.34-49) definite and DNA destroys at least a portion aminoacid sequence of the interactional cell protein of response gene product.Can be with the guidance of the aminoacid sequence that obtains as the oligonucleotide mixture that produces the gene order that can be used to screen the described cell protein of coding.For example, can finish screening by standard hybridization or round pcr.The technology that is used to prepare oligonucleotide mixture and screening be well known in the art (referring to, Ausubel for example, supra., and PCR Protocols:A Guide to Methods and Applications, 1990, Innis, M.et al., eds.Academic Press, Inc., New York).
In addition, can use the gene that causes while identification code and DNA to destroy the interactional cell protein of reactive protein.These methods comprise, for example, utilize the mode similar to the antibody Detection Techniques in known λ gt11 library to utilize DNA to destroy reactive protein, survey expression library with the DNA destruction reactive protein of mark.
Describe the interactional a kind of method in the proteic body that detects in detail, promptly two heterological systems, just in order to illustrate, rather than restriction.Described this system a kind of form (Chien etal., 1991, Proc.Natl.Acad.Sci.USA, 88:9578-9582), and can (Palo Alto CA) is purchased from Clontech.
In brief, adopt such system, made up the plasmid of the two kinds of hybrid proteins of encoding: a kind ofly form by combining the territory with DNA that DNA destroys the transcription activating protein that the response gene product merges, another kind of by with this plasmid of recombinating as the part in cDNA library in the activation domain of the transcription activating protein that merges of the agnoprotein of cDNA coding form.DNA is transformed into the Saccharomyces cerevisiae strain that contains reporter gene (as HBS or lacZ) in conjunction with territory fusion plasmid and cDNA library, and the regulatory region of described yeast strains contains the binding site of transcriptional activator.Any independent hybrid protein all can not activate transcribing of reporter gene: DNA in conjunction with the territory hybrid protein can not because it does not provide mobilizing function, the activation domain hybrid protein can not because it can not navigate to the binding site of activator.Functional activator has been rebuild in the interaction of two kinds of hybrid proteins, and causes the expression of reporter gene, and the mensuration by the reporter gene product can detect it.
Two heterological systems or relevant method can be used for screening and the interactional proteic activation domain of " bait " gene product library.Illustrate, but be not restriction, DNA destroys the response gene product can be used as the bait gene product.Total genome or cDNA sequence merge with the DNA of coding activation domain.The plasmid that combines the heterozygote that this library of merging in the territory and coding bait DNA destroy the response gene product with DNA in the strain of yeast report, is expressed those of reporter gene by cotransfection in the transformant that screening obtains.For example, but be not restriction, bait DNA destroys the response gene sequence, as the encoding sequence of DNA destruction response gene, can be cloned in the carrier, makes its translation be blended in the DNA of the proteic DNA of coding GAL4 in conjunction with the territory.These bacterium colonies of purifying separate and are responsible for the library plasmid that reporter gene is expressed.Identify by the plasmid-encoded albumen in library with dna sequencing then.
The cDNA library that can prepare clone with the conventional method of implementing in this area, from this library to destroy the interactional albumen of response gene product with DNA be to be detected.According to particular system described herein, for example, the cDNA fragment can be inserted carrier, make their translations be blended in the transcription activating domain of GAL4.Response gene-GAL4 fusion plasmid cotransformation can be destroyed in yeast strains with bait DNA in this library, and this yeast strains contains the lacZ gene by the promoters driven that comprises the GAL4 activation sequence.With DNA destroy the response gene product interactional, by the albumen that is blended in the GAL4 transcription activating domain of cDNA coding, with the active GAL4 albumen of reconstruct, and therefore drive the HIS3 expression of gene.Can detect the bacterium colony of expressing HIS3 by containing based on the growth on the Petri dish of the substratum of the shortage Histidine of semi-solid agar.Then can be from these bacterial strain purifying cDNA, and with the conventional technology production of implementing in this area with separates bait DNA destruction response gene-interaction protein.
Can determine that DNA destroys the interaction between response gene and the conditioning agent thereof by standard method well known in the art.
5.4.4. the method for screening reagent
The invention provides screening regulates the expression of DNA destruction reaction or regulates the interactional method that DNA destroys reaction and other albumen or molecule.
5.4.4.1. screening assay
Designed following mensuration, to identify some compounds, it is incorporated into DNA and destroys response gene or gene product, be incorporated into DNA and destroy interactional other cell protein of response gene product, be incorporated into and be subjected to DNA to destroy the cellular component that the response gene product influences, as albumen, or be incorporated into the interactional compound of interference DNA destruction response gene or gene product and other cell protein and regulate the compound that DNA destroys the activity (that is, regulating the level that DNA destruction response gene expression level and/or adjusting DNA destroy the response gene its lytic activity) of response gene.Can also utilize and identify that being incorporated into DNA destroys the mensuration that response gene is regulated the compound of sequence (as promoter sequence), referring to, Platt for example, K.A., 1994, J.Biol.Chem.269:28558-28562 is incorporated herein by reference in full at this, and described compound can be regulated the expression level that DNA destroys response gene.Described compound can include, but not limited to influence the little organic molecule that DNA destroys other expression of gene of response gene or some participation DNA destruction reaction paths, or other cell protein.The method of identifying described cell protein is described in above 5.4.3 joint.Described cell protein can participate in the Growth Inhibition of DNA disrupting agent and regulate.In addition, in these compounds, comprised that influencing DNA destroys the expression of response gene and/or the activity of DNA destruction response gene product, and can be used to regulate compound the Growth Inhibition resistance of DNA disrupting agent.
Described compound can include, but not limited to peptide, as soluble peptide, includes, but not limited to the member of the fusogenic peptide and the random peptide library of Ig tail; (referring to, Lam for example, K.S.et al., 1991, Nature 354:82-84; Houghten, R.et al., 1991, Nature 354:84-86), and combinatorial chemistry deutero-molecular library, described library by D-and/or L-configuration amino acid form, phospho-peptide (comprises, but be not limited at random or the phospho-peptide library part degeneracy, directed, referring to, Songyang for example, Z.etal., 1993, Cell 72:767-778), antibody (includes, but are not limited to polyclone, mono-clonal, humanization, antiidiotype, chimeric or single-chain antibody and FAb, F (ab ')
2With FAb expression library fragment and epi-position binding fragment thereof) and little organic or inorganic molecule.
Through being used for, for example, regulate the biological function that DNA destroys the response gene product, and be used to improve the Growth Inhibition resistance of DNA disrupting agent and/or the growth-inhibiting effect of enhancing DNA disrupting agent as mensuration compounds identified described herein.The 5.4.4.2. joint that is determined at hereinafter that is used for the validity of detection compound is discussed.
Vitro system can be designed, the compound that DNA of the present invention destroys the response gene product can be incorporated into to identify.Compounds identified can be used for, for example, the DNA that regulates wild-type and/or sudden change destroys the activity of response gene product, can be used to illustrate the biological function that DNA destroys the response gene product, can identify that destroying normal DNA destroys the interaction of response gene product, or use in the screening of the described interactional compound of autoclasia.
Be used for identifying that being incorporated into principle that DNA destroys the compound of response gene product is included in and is enough to make DNA destroy the response gene product and test compounds interacts and the reaction mixture of bonded condition and these two kinds of compositions of time preparation, form the mixture that in reaction mixture, to remove and/or to detect thus.Can adopt multiple mode to carry out described mensuration.For example, a kind of method of measuring comprises destroys the response gene product with DNA or test substances anchors on the solid phase, and detects the DNA that is anchored on the solid phase and destroy response gene product/test compounds mixture when reaction finishes.In described method in one embodiment, DNA can be destroyed the response gene product and be anchored on solid surface, and the direct or indirect mark test compounds of grappling not.
In practice, can easily microtiter plate be used as solid phase.Can be by non-covalent or covalent attachment, the one-tenth of grappling is separation-immobilized.Can finish non-covalent adhering to by also dry with the protein solution bag simply by solid surface.Perhaps, can be with treating the proteic specificity immobilized antibody of fixed, preferred monoclonal antibody anchors to solid surface with albumen.Can prepare surface and storage in advance.
In order to carry out this mensuration, loose composition is added the surface of the bag quilt of the composition that contains grappling.After reaction is finished, remain fixed at the alloy that forms remove under the condition of solid surface unreacted composition (as, by washing).Can adopt number of ways to finish the detection that is anchored on the mixture on the solid surface.When preliminary making during front loose composition, the detection that is fixed on lip-deep mark shows and has formed mixture.When not having the loose composition in preliminary making front, can detect with indirect labelling and be anchored on lip-deep mixture, for example, adopt the specific marker antibody (antibody can carry out direct mark or indirect labelling with the anti-Ig antibody of mark subsequently) of the loose composition in front.
Perhaps, can in liquid phase, react,, and detect mixture from unreacted component separating reaction product; For example, destroy the alloy that forms in the specificity immobilized antibody grappling solution of response gene product or test compounds, with the mixture of the specific marker antibody test grappling of other composition of possible mixture with DNA.
DNA destroy response gene or gene product can be in vivo with one or more cells in or extracellular molecules, as protein-interacting.Described molecule can include, but not limited to nucleic acid molecule and the albumen by the method evaluation of 5.4.3 joint description as mentioned.For the purpose of discussing, described molecule is called " binding partners " at this.The bonded compound that destroys DNA destruction response gene product can be used to regulate the activity that DNA destroys the response gene product.The bonded compound that destroys DNA destruction response gene product can be used to regulate the expression that DNA destroys response gene, for example by regulating the combination that DNA destroys the conditioning agent of response gene.Described compound can include, but are not limited to the peptide of all joints of 5.4.4.1 as mentioned description etc., and it can destroy the response gene product near DNA.
Be used for identifying and disturb DNA to destroy in response gene product and the cell thereof or the ultimate principle of the mensuration system of the interactional compound of extracellular binding partners is included in and is enough to make DNA destroy the response gene product and binding partners interacts and bonded condition and time preparation comprise the reaction mixture of these two kinds of compositions, form mixture thus.For the inhibition activity of test compounds, preparation feedback mixture under test compounds existence and non-existent condition.Test compounds can be included in the reaction mixture at first, or can add in the time after adding DNA destruction response gene product and binding partners thereof.Do not having under the condition of test compounds or with placebo incubation control reaction mixture.Detect the formation that DNA destroys alloy between response gene albumen and the binding partners then.In control reaction, form mixture, but in containing the reaction mixture of test compounds, do not form mixture, show that compound disturbs the interaction between DNA destruction response gene albumen and the interactional binding partners.In addition, the mixture in containing the proteic reaction mixture of test compounds and normal DNA destruction response gene forms, and also can compare with the mixture formation that contains in the proteic reaction mixture of test compounds and mutant DNA destruction response gene.Identify the destruction mutant at needs, but do not destroy under the situation of the proteic compound of normal DNA destruction response gene that this relatively is important.
Can disturb DNA to destroy the interactional compound of response gene product and binding partners thereof with heterogeneous or homogeneous phase form.Heterogeneous assays comprises destroys the response gene product with DNA or binding partners is anchored on the solid phase, and detects the mixture that is anchored on the solid phase when reaction finishes.In homogeneous determination, in liquid phase, carry out entire reaction.In any method, can change the order that adds reactant, to obtain different information about the compound of test.For example, can identify by the following method by for example competing the interactional test compounds of disturbing DNA to destroy between response gene product and the binding partners: in the presence of test substances, react, promptly by before adding DNA destruction reactive protein and interactional binding partners or simultaneously test substances is added reaction mixture.Perhaps,, can detect the test compounds of destroying preformed mixture, as the compound with higher binding constant of one of the composition of replacing mixture by after forming mixture, test compounds being added reaction mixture.Various forms has hereinafter briefly been described.
In the heterogeneous assays system, DNA is destroyed the response gene product or interactional binding partners is anchored on the solid phase, and the direct or indirect mark material of grappling not.In practice, can utilize microtiter plate easily.Can be by non-covalent or covalent attachment, substance fixed with grappling.Can finish non-covalent adhering to simply by destroying response gene product or binding partners solution bag with DNA by solid surface and dry.Perhaps, can material be anchored to solid surface with the specificity immobilized antibody for the treatment of the fixed material.Can prepare surface and storage in advance.
In order to carry out this mensuration, be with or without the surface that under the condition of test compounds the mating partner of fixed material is exposed to the bag quilt.After reaction is finished, remove unreacted composition (for example, by washing) and remain fixed in the mixture of any formation of solid surface.Can finish the detection of the mixture that is anchored on solid surface with multiple mode.When mark in advance during loose material, the detection that is fixed on lip-deep mark shows and has formed mixture.When not in advance during the loose material of mark, can detect with indirect labelling and be anchored on lip-deep mixture, for example, adopt the specific marker antibody (antibody can carry out direct mark or indirect labelling with the anti-Ig antibody of mark subsequently) of initial loose material.According to the order of adding reacted constituent, can detect and suppress the test compounds that mixture formed or destroyed preformed mixture.
Perhaps, can in liquid phase, react,, and detect mixture from unreacted component separating reaction product existing or not existing under the condition of test compounds; For example, the alloy that forms in the specificity immobilized antibody grappling solution with one of binding constituents is with the mixture of the specific marker antibody test grappling of other mating partner.Equally, according to adding the order of reagent, can identify the test compounds that suppresses mixture formation or destroy preformed mixture to liquid phase.
In a kind of alternate embodiment of the present invention, can use homogeneous determination.In the method, preparation DNA destroys the preformed mixture of response gene albumen and interactional binding partners, wherein mark DNA destroy response gene product or its binding partners, but because mixture forms, the signal that this mark produces by cancellation (referring to, the U.S. Patent No. 4,109 of Rubenstein for example, 496, it utilizes this method to carry out immunoassay).With the competition of one of material in the preformed mixture and replace the interpolation of the test substances of this material, will cause producing the signal that is higher than background.In this way, can identify that destroying DNA destroys the interactional test substances of reactive protein/binding partners.
In a kind of specific embodiment, can prepare DNA and destroy the response gene product, fix to adopt recombinant DNA technology.For example, can adopt fusion vector, as pGEX-5X-l, make DNA destroy reaction coding region and glutathione-S-transferase (GST) gene fusion, amalgamation mode makes and keeps it in conjunction with activity in the fusion rotein that obtains.Can the interactional binding partners of purifying, and be used to adopt the conventional method of implementing in this area to produce monoclonal antibody.For example, can use radio isotope by the conventional method of implementing in this area
125The I traget antibody.In heterogeneous assays, for example GST-DNA can be destroyed the reaction fusion rotein and be anchored to gsh-sepharose 4B.Then, can with allow to take place interact and the bonded mode test compounds exist or non-existent condition under the interactional binding partners of adding.When reaction finishes, can the unconjugated material of flush away, can monoclonal antibody adding system with mark in, and it is combined with the compound composition.Can keep and the associating radioactive amount of gsh-sepharose 4B by measuring, detection DNA destroys the interaction between response gene albumen and the interactional binding partners.Test compounds successfully suppresses interactional, and the radioactivity that will cause measuring reduces.
Perhaps, can under the condition that does not have solid gsh-sepharose 4B, GST-DNA destruction reaction fusion rotein and interactional binding partners be mixed together in the liquid.Can or add test compounds in these matter interactions afterwards.Then, this mixture can be added gsh-sepharose 4B, the unconjugated material of flush away.Equally, can detect the interactional inhibition degree of DNA destruction response gene product/binding partners with the associating radioactivity of pearl by antibody and the measurement that adds mark.
In another embodiment, can adopt the peptide fragment that destroys reactive protein and/or interactional binding partners corresponding to DNA to substitute in these two kinds of full-length proteins one or both and use these technology (is under the proteic situation at binding partners) in conjunction with the territory.Can use the method for the conventional any number implemented in this area to identify and the separation and combination site.These methods include, but not limited to the mutagenesis of gene of one of proteins encoded and screening coimmunoprecipitation measure in bonded destroy.Can select then to encode anaphragmic in the gene of second kind of material in the mixture.The proteic separately Gene Sequence Analysis of encoding will disclose corresponding to the proteic zone that participates in the interactional combination.Perhaps, can a kind of albumen be anchored to solid surface with method as described above in this section, and it is also combined with its binding partners interaction, described binding partners has been used proteolytic ferment, as trypsin treatment.After the washing, comprise that small peptide in conjunction with the mark in territory can keep and solid material associates, it can separate and identify by amino acid sequencing.Equally, in case obtained the gene of coding binding partners, can carry out the peptide fragment of through engineering approaches with expressing protein to short gene fragment, it is active to detect described segmental combination then, and purifying or synthetic.
For example, but not restriction, can be according to above description in this section, destroy the reaction fusion rotein and it is combined with the glutathione agarose pearl and DNA is destroyed gene product be anchored to solid material by preparation GST-DNA.Can use radio isotope,, and use proteolytic ferment, cut as trypsinase as the interactional binding partners of 35S mark.The GST-DNA that cleaved products can be added grappling then destroys the reaction fusion rotein, and makes in conjunction with taking place.Behind the unconjugated peptide of flush away, can represent the bond material of the mark in binding partner binds territory by the known method wash-out, purifying, and analysis of amino acid sequence.The peptide that produces evaluation like this be can synthesize, or recombinant DNA technology and suitable facilitation albumen fusion adopted.
5.4.4.2. the Growth Inhibition compound of DNA disrupting agent is regulated and/or is strengthened in screening
Can further screen the expression of adjusting DNA destruction response gene and/or interactional any reagent that DNA destroys reactive protein and its binding partners, destroy the adjusting and/or the Growth Inhibition abilities of enhancing DNA disrupting agent in cell such as antibody of reactive protein as 5.4.4.1 joint compounds identified, DNA.Any suitable propagation well known in the art or growth-inhibiting mensuration can be used for this purpose.In one embodiment, candidate agent and DNA disrupting agent are applied to the cell of clone, and definite Growth Inhibition changes.Preferably, determine that with the combination of the candidate agent of different concns and different concns DNA disrupting agent Growth Inhibition changes, so that determine to cause one or more combinations of the concentration of 50% candidate agent that suppresses and DNA disrupting agent, that is, and IC
50
In a kind of preferred embodiment, with the MTT proliferation assay (referring to, van deLoosdrechet for example, et al., 1994, J.Immunol.Methods 174:311-320; Ohnoet al., 1991, J.Immunol.Methods 145:199-203; Ferrari et al., 1990, J.Immunol.Methods 131:165-172; Alley et al., 1988, Cancer Res.48:589-601; Carmichael et al., 1987, Cancer Res.47:936-942; Gerlieret al., 1986, J.Immunol.Methods 65:55-63; Mosmann, 1983, J.Immunological Methods 65:55-63) screens with the combination of DNA disrupting agent and be used for cytostatic candidate agent.Candidate agent and DNA disrupting agent with selected concentration were handled cell 4-72 hour.The 3-of cell and appropriate amount (4,5-dimethylthiazole-2-yl)-2 then, 5-phenylbenzene tetrazolium bromide (MTT) incubation 1-8 hour together makes the cell of survival that MTT is converted into insoluble first
Cell in settling.After removing the excessive MTT that comprises in the supernatant liquor, add suitable MTT solvent, as DMSO solution, with the dissolving first
By determining the optical density(OD) at 570nm place, measure the proportional MTT concentration of number then with survivaling cell.Can measure the candidate agent of a large amount of different concns, so that determine to cause 50% candidate agent that suppresses and the concentration of DNA disrupting agent.
In another kind of preferred embodiment, use the alamarBlue of cell proliferation
TMMeasure screen can be used for cytostatic one or more candidate agents (referring to, Page etal. for example, 1993, Int.J.Oncol.3:473-476).AlamarBlue
TMMeasure and measure cellular respiration, and measuring used as viable cell number.The interior environment of proliferative cell has higher reductibility than the interior environment of non-proliferative cell.For example, in breeding, NADPH/NADP, FADH/FAD, the ratio of FMNH/FMN and NADH/NAF increases.These metabolic intermediates can reduce alamarBlue, therefore, can be used to monitor cell proliferation.Cell number in the sample of the processing of measuring by alamarBlue can be expressed as the per-cent with respect to the cell number in the untreated control sample.
In a kind of particular, carry out alamarBlue
TMMeasure, decide the transfection titre curve of siRNA that DNA destroys response gene whether owing to select the DNA disrupting agent of concentration, change as the existence of camptothecine to determine target.Decide the siRNA transfectional cell that DNA destroys response gene with target.After the siRNA transfection 4 hours, under the condition that has or do not exist the DNA disrupting agent, add the DMEM/10% foetal calf serum in 100 microlitres/hole, with plate incubation 68 hours under the condition of 37 ℃ and 5%CO2.From the hole, remove substratum, and with containing 10% (vol/vol) alamarBlue
TMReagent (Biosource International Inc., Camarillo, CA) and the DMEM/10% foetal calf serum (Invitrogen) of the 1M Hepes damping fluid tissue culture reagent (Invitrogen) of 0.001 volume replace.With plate 37 ℃ of incubations 2 hours, adopt then Softmax Pro 3.1.2 software (MolecularDevices) SpectraMax plus read the plate device (Molecular Devices, Sunnyvale, CA) on, 570 and the 600nm wavelength readings.Decide the reduction per-cent in hole of the siRNA transfection that DNA destruction reacts and the hole of luciferase siRNA transfection compares with existing or do not exist under the condition of DNA disrupting agent with the target of certain titre.The % reduction numerical value that when not having the DNA disrupting agent hole of 0nM luciferase siRNA transfection is calculated is considered to 100%.
5.4.4.3. compounds identified
Compounds identified comprises in this screening has proved that DNA destroys the compound that the Growth Inhibition ability of DNA disrupting agent was expressed and regulated and/or strengthened to response gene in the selectivity adjusting cell.These compounds include, but not limited to siRNA, antisense molecule, ribozyme, triple helical, antibody and peptide molecule, aptamrs and little organic or inorganic molecule.
Compounds identified comprises that also regulating DNA destroys the interactional compound that reacts with other albumen or molecule in this screening.In one embodiment, compounds identified is to regulate the interactional compound that DNA destroys reactive protein and its interaction mating partner in this screening.In another embodiment, compounds identified is to regulate the interactional compound that DNA destroys response gene and transcriptional regulatory agent in this screening.
5.4.5. diagnosis
Several different methods can be used to diagnose with the prognostic assessment because DNA destroys cell that the adjusting defective of reaction causes to the DNA disrupting agent, as the Growth Inhibition resistance of camptothecine be used to identify to have the experimenter who the growth-inhibiting effect of DNA disrupting agent is had the tendency of resistance.
In one embodiment, this method comprises determines that DNA destroys the response gene expression level in the cell, and the expression level that wherein is higher than predetermined threshold level shows that cell has DNA disrupting agent resistance.Preferably, predetermined threshold level is at least 2 times, 4 times, 8 times or 10 times of the DNA normal expression level of destroying response gene.In another embodiment, the invention provides the method for the DNA disrupting agent resistance in the assessment cell, comprise the abundance level of determining to be destroyed by DNA in the cell response gene encoded protein, the abundance water-glass clear-cells that wherein is higher than predetermined threshold level has DNA disrupting agent resistance.In another embodiment, the invention provides the method for the DNA disrupting agent resistance in the assessment cell, comprise the activity level of determining to be destroyed by DNA in the mammalian cell response gene encoded protein, the activity level that wherein is higher than predetermined threshold level shows that cell has DNA disrupting agent resistance.Preferably, abundance or active predetermined threshold level are that DNA destroys the normal abundance of reactive protein or at least 2 times, 4 times, 8 times or 10 times of activity level.
Described method is passable, for example, uses reagent such as DNA destruction response gene nucleotide sequence and at the interaction gene product, comprises the antibody of its peptide fragment.Particularly, described reagent can be used for, and for example (1) is detected DNA and destroyed the existence that suddenlys change in the response gene, or the mRNA of detection DNA destruction response gene is not enough with respect to the overexpression or the expression of normal expression level; (2) detect DNA and destroy the response gene product destroys the reactive protein normal level with respect to DNA excess abundance or abundance deficiency.
For example, can comprise at least a specific DNA described herein by use destroys the test kit that response gene nucleic acid or anti-DNA destroy reaginic antibody reagent and carries out method described herein, described method can be advantageously used in for example clinical the setting, has with DNA with diagnosis and destroys relevant illness of response gene or unusual patient.
Destroy the reaction sudden change in order to detect DNA, the cell of any tool nuclear can be used as the initial source of genomic nucleic acids.In order to detect expression or the DNA destruction response gene product that DNA destroys response gene, can use any cell type or the tissue of having expressed DNA destruction response gene.
Detection technique based on nucleic acid is described in hereinafter 5.4.5.1 joint.The peptide detection technique is described in hereinafter 5.4.5.2 joint.
5.4.5.1.DNA destroy the detection that response gene is expressed
Can utilize the expression of DNA destruction response gene in many technology for detection cell or tissues, for example DNA destroys the cell levels of response gene transcript and/or the existence or the shortage of sudden change.Can will be used as the starting point of described determination techniques from the nucleic acid of any tool karyocyte, and can be according to well known to a person skilled in the art that the standard nucleic acid preparation procedure separates.For example, can use all to comprise the expression level that DNA destroys one or more polynucleotide probes measurements DNA destruction response gene of the nucleotide sequence in the response gene, determine that DNA destroys the expression level of response gene.In particularly preferred embodiment of the present invention, this method is used for the resistance of diagnosing human cancer to the treatment of employing DNA disrupting agent.
The hybridization or the amplification assay that DNA can be used for biological sample relate to structure unusual that DNA destroys response gene with detection, comprise point mutation, insertion, disappearance and chromosome rearrangement.Described mensuration can include, but not limited to Southern and analyze single-strand conformation polymorphism analysis (SSCP), dna microarray analysis and pcr analysis.
Being used to detect the described diagnostic method that DNA destroys the response gene specific mutant can comprise, for example, contact and incubation comprise the nucleic acid of recombinant DNA molecules, cloned genes or its degeneracy variant, it is available from sample, as patient's sample or other the suitable cell source derived from the nucleic acid reagent with one or more marks that comprise recombinant DNA molecules, cloned genes or its degeneracy variant, the conditions favouring of contact and incubation carries out specificity annealing with its complementary sequence in these reagent and DNA destruction response gene.Preferably, the length of these nucleic acid reagents is 15-30 Nucleotide at least.Behind the incubation, all unannealed nucleic acid are removed from nucleic acid: DNA destruction reaction molecular crossbred.Detect exist (if having any described molecule) of the nucleic acid carried out hybridization then.Adopt described detection scheme, the nucleic acid from purpose cell type or tissue can be fixed on the solid support of film for example, or be fixed on the frosting on the surface of microtiter plate for example or polystyrene bead.In this case, behind incubation, can easily the nucleic acid reagent of unannealed mark be removed.With well known to a person skilled in the art standard technique, the DNA that has finished remaining, annealed, mark destroys the detection of reaction nucleic acid reagent.Destroying response gene with nucleic acid reagent annealed DNA can compare with the annealing pattern of destroying the expectation of response gene sequence from normal DNA, so that determine whether to exist DNA to destroy the response gene sudden change.
The alternative diagnostic methods that the DNA that is used for detecting patient's sample or other suitable cell source destroys response gene specific nucleic acid molecule can comprise their amplification, for example pass through pcr amplification (at Mullis, K.B., 1987, U.S. Patent No. 4,683,202), then with the molecule that well known to a person skilled in the art the technology for detection amplification.Desired those compare under the situation that the DNA of normal copy destroys response gene if can be with the extension increasing sequence that obtains only contain during with nucleic acid amplification, so that determine whether to exist DNA to destroy the response gene sudden change.
Destroy in the nucleotide sequence of response gene in described hybridization and/or the preferred DNA of pcr analysis, comprise those that detect DNA destruction response gene splice site sudden change existence.
In addition, can carry out known gene type assay technology, carry the individuality that DNA destroys the response gene sudden change with evaluation.Described technology comprises, for example, uses restrictive fragment length polymerphism (RFLPs), and it comprises the sequence variations in one of the recognition site of specificity restriction enzyme of use.
In addition, described the method for analyzing the dna polymorphism that can be used for identification of dna destruction reaction sudden change, described method is utilized the existence of the short polyphone reiterated DNA sequences of different numbers between the restriction enzyme sites.For example Weber (U.S. Patent No. 5,075,217 is introduced the document as a reference in full at this) has described the dna marker thing based on length polymorphism in the short polyphone of (dC-dA) n-(dG-dT) n tumor-necrosis factor glycoproteins block.The equipartition of estimating (dC-dA) n-(dG-dT) n block is 30,000-60,000bp.Closely adjacent marker shows high frequency heredity altogether on the space, and particularly useful to identifying such as the transgenation of the sudden change in the DNA destruction response gene disease and the illness relevant with DNA destruction reaction sudden change with diagnosis.
Caskey etc. (U.S. Patent No. 5,364,759 is introduced the document as a reference in full at this) have described the DNA spectrum analysis that is used to detect short three and TTTC.This method comprises the extraction target DNA, destroys response gene as DNA, the DNA that amplification is extracted, and the mark tumor-necrosis factor glycoproteins is to form the genotype collection of illustrative plates of individual DNA.
Also can measure the expression level that DNA destroys response gene.For example, can separate and utilize the check of hybridization described above or round pcr known or suspect that expressible dna destroys the cell type or the tissue of response gene, as the RNA of the cancer cell type of performance DNA disrupting agent resistance.Isolated cells can be derived from cell culture or patient.The analysis of taking from the cell of culture can be the part that is used as based on the gene therapy technology of cell, or the check compound is to the steps necessary in the cell assessment of the effect of DNA destruction response gene expression.Described analysis can disclose the quantitative and qualitative aspect that DNA destroys the expression pattern of response gene, comprises that DNA destroys activation or deactivation that response gene is expressed.
In a kind of embodiment of described detection scheme, from purpose RNA molecule synthesis cDNA molecule (as, by being cDNA with RNA molecule reverse transcription).Then the sequence in the cDNA is used as nucleic acid amplification reaction, as the template of uses such as pcr amplification reaction.Be selected from DNA destruction response gene nucleic acid reagent as the reverse transcription of this method and the nucleic acid reagent of the synthetic initial reagent (as primer) in the nucleic acid amplification step.The preferred length of described nucleic acid reagent is 9-30 Nucleotide at least.In order to detect amplified production, can carry out nucleic acid amplification with radioactivity or nonradioactive labeling's Nucleotide.Perhaps, can prepare enough amplified productions, make and can pass through to use any suitable nucleic acid staining method, as, product shown by the standard ethidium bromide staining.
In addition, can " original position " carry out described DNA and destroy response gene and express and measure, that is, directly the tissue slice of the patient tissue that obtains from biopsy thing or excision thing (fixing and/refrigerated) be measured, thereby needn't be carried out nucleic acid purification.The nucleic acid that DNA can be destroyed response gene as the probe in the described original position program and/or primer (referring to, Nuovo for example, G.J., 1992, " PCR In Situ Hybridization:Protocols And Applications ", Raven Press, NY).
Perhaps,, can carry out standard Northern and analyze, destroy the mRNA expression level of response gene to determine DNA if can obtain the suitable cell of capacity.
Also can destroy response gene and express, destroy the cell levels of reaction transcript and/or the existence or the shortage of sudden change as DNA with the DNA in the dna microarray technical evaluation cell or tissue.In described technology, one or more polynucleotide probes that all comprise the sequence of DNA destruction response gene can be used for the expression that monitoring of DNA is destroyed response gene.Therefore, the invention provides the dna microarray that comprises polynucleotide probes, described probe comprises the sequence that DNA destroys response gene.
Any type of dna microarray technology can be used in combination with the present invention.In one embodiment, by DNA being destroyed the cDNA fragment of response gene, be deposited on the suitable surface as the PCR product of full-length cDNA, ESTs etc., preparation spot cDNA microarray (referring to, DeRisi et al. for example, 1996, Nature Genetics 14:457-460; Shalon etal., 1996, Genome Res.6:689-645; Schena et al., 1995, Proc.Natl.Acad.Sci.U.S.A.93:10539-11286; With Duggan et al., Nature GeneticsSupplement 21:10-14).In another embodiment, by photoetching technique from the teeth outwards original position synthetic contain with DNA destroy the sequence complementary oligonucleotide of response gene high density oligonucleotide array (referring to, for example, Fodor et al., 1991, Science 251:767-773; Pease et al., 1994, Proc.Natl.Acad.Sci.U.S.A.91:5022-5026; Lockhart et al., 1996, Nature Biotechnology 14:1675; McGall et al., 1996, Proc.Natl.Acad.Sci.U.S.A.93:13555-13560; United States Patent(USP) Nos. 5,578,832; 5,556,752; 5,510,270; 5,858,659; With 6,040,138).The microarray technology of this form to detect single nucleotide polymorphism (SNPs) particularly useful (referring to, Hacia et al. for example, 1999, Nat Genet.22:164-7; Wang et al., 1998, Science280:1077-82).In another embodiment, by ink-jet technology from the teeth outwards original position synthetic contain with DNA destroy the sequence complementary oligonucleotide of response gene high density oligonucleotide array (referring to, Blanchard for example, the open WO 98/41531 of disclosed international patent application on September 24th, 1998; Blanchard et al., 1996, Biosensors andBioelectronics 11:687-690; Blanchard, 1998, in Synthetic DNA Arraysin Genetic Engineering, Vol.20, J.K.Setlow, Ed., Plenum Press, New York at pages 111-123).In another embodiment, the dna microarray that allows to carry out the control of electronics severity can be used in combination with the polynucleotide probes of the sequence that comprises DNA destruction response gene (referring to, for example U.S. Patent No. 5,849, and 486).
5.4.5.2.DNA destroy the detection of response gene product
Can be according to description herein, the DNA of anti-wild-type or sudden change is destroyed diagnosis and the prognosis agent of the antibody of response gene product or its conservative variant or peptide fragment as DNA disrupting agent resistance.Described diagnostic method can be used to detect expression level unusual that DNA destroys response gene, or DNA destroys time, tissue, cell or Subcellular Localization unusual of the structure of response gene product and/or product.
Because it is intracellular gene product that DNA destroys the response gene product, antibody described below and method of immunity destroy the illness that the adjusting defective of response gene causes at assessment treatment DNA, have importance in the external application as the effect of proliferative disease.Antibody as mentioned below or antibody fragment can be used for the possible treatment compound of in-vitro screening, to determine them DNA are destroyed the effect that response gene is expressed and DNA destruction reaction peptide is produced.Can identify that DNA is destroyed the relevant illness of adjusting defective of reacting has the compound of beneficial effect, and determine the treatment effective dose.
Also can use for example external method of immunity to assess the effectiveness of DNA being destroyed the relevant illness of the adjusting defective of reaction based on the gene therapy of cell.The antibody that can external use anti-DNA destroys the reaction peptide is determined to destroy the DNA that finishes in the cell of reaction peptide and destroy the response gene expression level to produce DNA genetically engineered.Evidence disclosed herein shows that it is gene product in the cell that DNA destroys the response gene product, and described mensuration is preferably carried out with cell lysate or extract.Described analysis makes it possible to determine to obtain the necessary transformant number of interior curative effect, and the optimized gene replacement scheme.
Tissue to be analyzed or cell type generally include known or suspect that expressible dna destroys those of response gene, as, DNA disrupting agent resistance cancer cell type.The protein separating method of Shi Yonging can be herein, for example, those that describe among Harlow and the Lane (Harlow, E.and Lane, D., 1988, " Antibodies:A Laboratory Manual ", ColdSpring Harbor Laboratory Press, Cold Spring Harbor, New York), be incorporated herein by reference in full at this.Isolated cells can be derived from cell culture or patient.The analysis of taking from the cell of culture can be used to check compound that DNA is destroyed the effect that response gene is expressed.
Being used to detect the preferred diagnostic method that DNA destroys response gene product or its conservative variant or peptide fragment can comprise, for example immunoassay, wherein by DNA destroy response gene product or its conservative variant or peptide fragment and anti-DNA destruction response gene product specific antibody interaction partners its detect.
For example, antibody or the antibody fragment that destroys reactive protein in conjunction with DNA can be used for the existence of DNA destruction response gene product or its conservative variant or peptide fragment is carried out quantitatively or qualitative detection.This can (referring to this joint hereinafter) immunofluorescence technique be finished by for example use and opticmicroscope, flow cytometry or the fluorescently-labeled antibody of fluoroscopic examination bonded.If described DNA destroys the response gene product at cell surface expression, described technology is particularly preferred.
Be used for antibody of the present invention (or its fragment) and can additionally carry out the histology use, as be used for immunofluorescence or immunoelectron microscope, be used for the in situ detection that DNA destroys response gene product or its conservative variant or peptide fragment.Can be by removing histological specimen from the patient, and, finish in situ detection with its antibody that is applied to mark of the present invention.Preferably cover on the biological sample and administration of antibodies (or fragment) by antibody (or fragment) with mark.By using described program, can determine that not only DNA destroys the existence of response gene product or its conservative variant or peptide fragment, also can determine its distribution in being examined tissue.Employing the present invention those skilled in the art will readily recognize that, can improve in the multiple Histological method (as dyeing procedure) any one, to finish described in situ detection.
The immunoassay that DNA destroys response gene product or its conservative variant or peptide fragment are usually included in the antibody that can identification of dna destroys the detectability mark of response gene product or its conservative variant or peptide fragment and have incubation sample down, as biofluid, tissue extract, the cell of new results or the lysate of the cell that incubation is crossed in cell culture, and by any technology for detection bonded antibody well known in the art.
Can make biological sample contact and be fixed on solid support or carrier, as nitrocellulose or other can fixed cell, on the solid support of cell granulations or soluble protein.Can handle with the DNA destruction reactive protein specific antibody of detectability mark subsequently with suitable damping fluid washing upholder then.Can wash solid support to remove unconjugated antibody with damping fluid for the second time then.Can detect the amount of the bonded marker on the solid support then by ordinary method.
" solid support or carrier " is intended to represent can conjugated antigen or any upholder of antibody.Known upholder or carrier comprise glass, polystyrene, polypropylene glycol, polyoxyethylene glycol, dextran, nylon, amylase, natural and Mierocrystalline cellulose, polyacrylamide, gabbro and the magnetite modified.The character of carrier can be soluble to a certain degree or insoluble, to be used for purpose of the present invention.Support material can have any possible node configuration substantially, as long as the link coupled molecule can conjugated antigen or antibody.Therefore, the upholder configuration can be a spheric, as pearl or cylindrical, as is positioned at the outside surface of test trough internal surface or bar.Perhaps, the surface can be flat, as sheet, test strip etc.Preferred upholder comprises polystyrene bead.Those skilled in the art will know that to be used for binding antibody or antigenic many other suitable carriers, maybe can determine by using normal experiment.
Can determine that given batch anti-DNA destroys the combination activity of response gene product antibody according to known method.Those skilled in the art can determine each operating and optimum condition determination of measuring by normal experiment.
One of approach that can the detectability marker DNA destroys response gene peptide specific antibody is by it is connected with enzyme, and be used for enzyme immunoassay (EIA) (Voller, A., " TheEnzyme Linked Immunosorbent Assay (ELISA) ", 1978, DiagnosticHorizons 2:1-7, Microbiological Associates Quarterly Publication, Walkersville, MD); Voller, A.et al., 1978, J.Clin.Pathol.31:507-520; Butler, J.E., 1981, Meth.Enzymol.73:482-523; Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, FL; Ishikawa, E.etal., (eds.), and 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo).With the enzyme of antibodies will with suitable substrate, preferred chromophoric substrate reaction, reactive mode makes that generation can be by spectrophotometric for example, fluorescent method or the chemical part by the detection of visual inspection method.The enzyme that can be used to detection property traget antibody comprises, but be not limited to malate dehydrogenase (malic acid dehydrogenase), staphylococcal nuclease, Δ-5-steroidal class isomerase, Alcohol Dehydrogenase from Yeast, alpha-phosphate glyceryl ester, desaturase, triosephosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase enzymes, rnase, urease, catalase, glucose-6-phosphate dehydrogenase (G6PD), glucoamylase and acetylcholinesterase.The colorimetry of chromophoric substrate that can be by using enzyme is finished detection.Also can finish this detection by the enzymatic reaction degree of naked eyes comparison to the substrate of the standard substance of similar preparation.
Also can finish detection with in multiple other immunoassay any one.For example, by radiolabelled antibody or antibody fragment, can by use radioimmunoassay (RIA) detect DNA destroy the reaction peptide (referring to, Weintraub for example, B., Principles ofRadioimmunoassays, Seventh Training Course on Radioligand AssayTechniques, The Endocrine Society, March 1986, are hereby incorporated by).Can be by such as using gamma counter or scintillometer or by autoradiographic method detection of radioactive isotropic substance.
Also can use the fluorescent chemicals traget antibody.When fluorescently-labeled antibody is exposed to the light time with suitable wavelength, then because fluorescence can detect its existence.In most of normally used fluorescently-labeled compounds, comprise fluorescein isothiocyanate, rhodamine, phycoerythrin, Phycocyanins, C-, allophycocyanin, Phthalyldicarboxaldehyde and fluorescamine.
Also can use the fluorescent emission metal, as
152Eu or other lanthanide series metal detectability traget antibody.Can use metal-chelating group that these metals are connected with antibody such as Diethylenetriamine valeric acid (DTPA) or ethylenediamine tetraacetic acid (EDTA) (EDTA).
Also can be by antibody and chemiluminescence compound coupling are detected antibody.Determine the existence of the antibody of chemiluminescent labeling then by the existence that detects the fluorescence that in chemical reaction process, produces.The example of useful especially chemiluminescent labeling compound is luminol,3-aminophthalic acid cyclic hydrazide, different luminol,3-aminophthalic acid cyclic hydrazide, heromatic acridinium ester, imidazoles, acridinium salt and barkite.
Equally, can use bioluminescent compound mark antibody of the present invention.Noclilucence is a class chemoluminescence of finding in the biosystem, and wherein catalytic protein has increased the efficient of chemiluminescence reaction.By detecting luminous existence, determine the existence of bioluminescent protein.The important biomolecule luminophor that is used for the mark purpose is fluorescein, luciferase and aequorin.
Destroy the method that response gene is expressed 5.4.6. regulate DNA
Can use and regulate the expression that DNA destroys response gene in the multiple therapy methods body according to the present invention.For example, can be to the siRNA molecular engineeringization, and be used for making in vivo DNA to destroy the response gene silence.Also can carry out through engineering approaches, and be used for the translation of blocking dna destruction reaction mRNA in the body the antisense DNA molecule.Perhaps, can design ribozyme molecule, destroy reaction mRNA with cutting in the body and destruction DNA.In another kind of scheme, design oligonucleotides is distinguished (district that comprises the encoding sequence upstream) hybridization so that destroy 5 ' of response gene with DNA, and formation can be used to block or reduce the triple-helix structure that DNA destruction response gene is transcribed.If desired, also can design oligonucleotides so that with the binding site hybridization of negative conditioning agent with form triple-helix structure, so that the combining and strengthen transcribing of DNA destruction response gene of blocking-up and negative conditioning agent.
In a kind of preferred embodiment, design siRNA, antisense molecule, ribozyme and triple helical Nucleotide to be suppressing one or more DNA and destroy the translation of reaction isoforms or to transcribe, and to other may to destroy other expression of gene influences of the total one or more motifs of response gene minimum with DNA.For reaching this purpose, should destroy the distinctive correlated series of response gene based on DNA and design employed oligonucleotide.
For example, but be not restriction, oligonucleotide should not drop on DNA and destroy the nucleotide sequence of response gene and the highest zone of nucleotide sequence homology of its gene.Under the situation of antisense molecule, described sequence preference is selected from tabulation above.The length of sequence is at least 18 Nucleotide preferably, so that the enough strong annealing of realization and said target mrna sequence, thereby the translation of prevention sequence.Izant?et?al.,1984,Cell,36:1007-1015;Rosenberg?et?al.,1985,Nature,313:703-706。
Under the situation of " tup " type ribozyme, the target sequence of ribozyme is preferably selected from tabulation above.Ribozyme is the active RNA molecule of endonuclease with high special.Hammerhead ribozyme comprises at least a portion complementary hybridization region of nucleotide sequence and target RNA and is fit to the catalytic domain of cutting target RNA.Hybridization region comprises nine (9) individual or more a plurality of Nucleotide.Therefore, hammerhead ribozyme of the present invention has and sequence complementary hybridization region listed above, and length is at least 9 Nucleotide.The structure of described ribozyme and production are well known in the art, and are described in Haseloff et al., 1988, Nature, 334:585-591 more completely.
Ribozyme of the present invention also comprises RNA endonuclease (after this being called " Cech type ribozyme "), as natural existence the in Tetrahymena Thermophila (being called IVS or L-19IVS RNA), and the ribozyme (Zaug that is fully described by Thomas Cech and co-worker thereof, et al., 1984, Science, 224:574-578; Zaug and Cech, 1986, Science, 231:470-475; Zaug, et al., 1986, Nature, 324:429-433; The disclosed international patent application No.WO88/04300 of UniversityPatents Inc.; Been et al., 1986, Cell, 47:207-216).The Cech endonuclease has 8 base pair avtive spots with target RNA sequence hybridization, and the cutting of target RNA is taking place thereafter.
For with DNA destroy response gene 5 ' terminal hybridization and with its formation triple-helix structure, and can be used to block the oligonucleotide of transcribing, they preferably destroy the sequence complementation of 5 ' end of non-existent DNA destruction response gene in the reacting phase correlation gene with impregnable other DNA of expression level.Described sequence preferably do not comprise yet DNA destroy in the reaction promotor in addition slightly with the zone of described other dna homolog.Can use aforesaid compound by several different methods well known in the art, include, but not limited to use liposome as delivery vectors.When being in anti-degraded form, also can use naked DNA or RNA molecule, described anti-degraded is by modifying end, by forming ring molecule, or by using alternating bond, comprises the key that phosphorothioate bond and thiophosphoryl are modified.In addition, can pass nucleic acid, wherein nucleic acid molecule be puted together in polylysine or Transferrins,iron complexes by promoting transhipment to send.Also can pass through multiple virus vector, include, but are not limited in retrovirus, vaccinia virus, AAV and the adenovirus any one, with nucleic acid delivery in cell.
Perhaps, can make up the recombinant nucleic acid molecules of encoding or destroying the response gene nucleic acid molecule as described antisense molecule, ribozyme, triple helical or DNA.This nucleic acid molecule can be RNA or DNA.If nucleic acid encoding RNA, this sequence preference is connected with the regulatory element operability, so that produce enough RNA products of the needs of copy.Regulatory element can allow the composing type of sequence or adjustment type to transcribe.Can be in vivo, that is, the transfer vector of one or more RNA that in the cell of biology, will encode, as bacterial plasmid or viral RNA or DNA transfection in cell.(as, Llewellyn et al., 1987, J.Mol.Biol., 195:115-123; Hanahanet al.1983, J.Mol.Biol., 166:557-580).In case be positioned at cell, transfer vector can duplicate, and is transcribed by the cell aggregation enzyme, to produce RNA, perhaps can be integrated into the genome of host cell.Perhaps, can pass through the micromanipulation technology, as micro-injection, the transfer vector transfection of sequence that will comprise one or more RNA that encode makes transfer vector or its be partially integrated in the genome of host cell in cell or transfered cell.
RNAi also can be used for the expression that knockout dna destroys response gene.In one embodiment, be used to the mRNA that degrades with the double stranded rna molecule of 21-23 Nucleotide of the homologous region hybridization that destroys the mRNA that response gene transcribes from DNA, thereby make the expression " silence " of DNA destruction response gene.Preferably, dsRNA has hybridization region, as, the double stranded region of 19 Nucleotide, itself and DNA destroy the sequence complementation of the encoding sequence of response gene.Any target can be decided DNA and destroy response gene, be used for the present invention as the siRNA of the suitable encoding sequence of human STK6 or TPX2 gene.As exemplary embodiment, according to the Standard Selection rule (referring to, Elbashir et al. for example, 2002, Methods 26:199-213 is incorporated herein by reference in full at this) the design target decide the double-stranded siRNA of 21 Nucleotide of the coding region of DNA destruction response gene.
Can use any standard method that is used to import siRNA.In one embodiment, the induced gene silence by the siRNA that decides DNA destruction response gene to the presented by cells target (referring to, for example, Elbashir et al., 2001, Nature 411,494-498; Elbashir et al., 2001, Genes Dev.15,188-200 introduces all above-mentioned documents as a reference in full at this).SiRNA can chemosynthesis, perhaps derived from by the cutting of reorganization nickase to double-stranded RNA.Being used to import the another kind of method that makes DNA destroy the double-stranded DNA (dsRNA) of response gene silence is shRNA, that is, short hairpin RNA (referring to, Paddison et al. for example, 2002, Genes Dev.16,948-958; Brummelkamp et al., 2002, Science296,550-553; Sui, G.et al.2002, Proc.Natl.Acad.Sci.USA 99,5515-5520 introduces all above-mentioned documents as a reference in full at this).In the method, express target from plasmid (or virus) and decide the siRNA that DNA destroys response gene, it is as inverted repeats, has the ring sequence that interleaves with the formation hairpin structure.The rna transcription thing that contains hair clip that obtains by nickase processing is used for reticent siRNA with generation subsequently.Can be in cell stably express based on the shRNA of plasmid, make and can in cell, carry out long-term gene silencing in vitro and in vivo (referring to, McCaffrey et al.2002, Nature 418,38-39; Xia et al., 2002, Nat.Biotech.20,1006-1010; Lewis et al., 2002, Nat.Genetics 32,107-108; Rubinson et al., 2003, Nat.Genetics 33,401-406; Tiscornia et al., 2003, Proc.AM.Acad.Sci.USA100,1844-1848 introduces all above-mentioned documents as a reference in full at this).Also target can be decided to send in the siRNA body that DNA destroys response gene to be delivered to Mammals, in the organ or tissue as the mankind (referring to, Song et al.2003 for example, Nat.Medicine 9,347-351; Sorensen etal., 2003, J.Mol.Biol.327,761-766; Lewis et al., 2002, Nat.Genetics32,107-108 introduces all above-mentioned documents as a reference in full at this).In the method, with the solution intravenous injection of siRNA in Mammals.SiRNA can arrive purpose organ or tissue then, and effectively reduces target gene expression in mammalian organs or the tissue.
5.4.7. regulate the method that DNA destroys active and/or its approach of reactive protein
Can destroy the activity that DNA destruction reactive protein is regulated in the interaction between reactive protein and its binding partners by regulating DNA.In one embodiment, can use reagent, as the combination of binding partners as described in antibody, aptamers, the little organic or inorganic molecules in inhibiting, so that regulate DNA disrupting agent resistance.In another embodiment, can use reagent, destroy proteic activity in the reactive protein adjusting approach as antibody, aptamers, little organic or inorganic molecules in inhibiting DNA, so that regulate DNA disrupting agent resistance.In one embodiment, use kinase inhibitor, regulate DNA as Herbimycin, Gleevec, Genistein, Lavendustin, Iressa and destroy the kinase whose activity of reactive protein.
5.4.8. decide by target that DNA destroys response gene and/or its product carries out cancer therapy
Can destroy that reaction is expressed and/or active method and/or composition and the combination therapy of DNA disrupting agent suffer from the patient of cancer with adjusting mentioned above DNA.Particularly, described method and/or composition and DNA disrupting agent can be united use, be used for the treatment of to suffer from and show the patient of cancer that DNA destroys the DNA disrupting agent resistance of reaction mediation.Described therapy can be used for the treatment of cancer, includes, but not limited to rhabdosarcoma, neuroblastoma and glioblastoma, small cell lung cancer, osteosarcoma, carcinoma of the pancreas, mammary gland and prostate cancer, mouse melanoma and leukemia, and B cell lymphoma.
In preferred embodiments, method of the present invention and/or composition and DNA disrupting agent are united the patient who is used for the treatment of the cancer of suffering from the DNA disrupting agent resistance that shows DNA destruction reaction mediation.In described embodiment, regulate that DNA destroys that reaction is expressed and/or active, giving the susceptibility of cancer cells, thereby give or strengthen the validity of DNA disrupting agent treatment to the DNA disrupting agent.
In combination therapy, can be before using the DNA disrupting agent, simultaneously or use one or more compositions of the present invention afterwards.In one embodiment, before using the DNA disrupting agent, use composition of the present invention.Can determine to use timed interval between composition of the present invention and the DNA disrupting agent by the normal experiment that those skilled in the art are familiar with.In one embodiment, after reaching the threshold value that needs, DNA destruction reactive protein level gives DNA disrupting agent.Can determine that DNA destroys the level of reactive protein by adopting above-described any technology.
In another embodiment, use composition of the present invention simultaneously with the DNA disrupting agent.
In another embodiment, also after using the DNA disrupting agent, use one or more compositions of the present invention.When one or more compositions of the present invention that the long half time of DNA disrupting agent uses in treatment, described using can be useful especially.
It will be understood by those skilled in the art that the arbitrary combination of the different administration time that to use composition of the present invention and DNA disrupting agent.For example, when the long half time of DNA disrupting agent during, preferably before or after using the DNA disrupting agent, use composition of the present invention in composition of the present invention.
Use the frequency of composition of the present invention or depend on that at interval the DNA of needs destroys reaction level, can determine this level by above-described any technology.Change into the level that is higher or lower than needs when DNA destroys the reactive protein level, can increase or reduce the frequency of administration of composition of the present invention.
Can by any method assessment well known in the art separately or with the effect or the benefit of the co-administered composition of the present invention of DNA disrupting agent, for example, by requiring based on the dosage of measuring survival rate, side effect, DNA disrupting agent or the method for its arbitrary combination.If being applied in of composition of the present invention realized any one or multiple benefit among the patient, as increase survival rate, reduce side effect, reduce the dosage requirement of DNA disrupting agent, think that then composition of the present invention strengthened DNA disrupting agent treatment, and think that this method is effective.
5.5. pharmaceutical preparation and route of administration
Can use to the patient with the treatment effective dose and determine to influence STK6 genetic expression or the active compound of gene product, regulate the relevant illness of defective with STK6 with treatment or improvement.The treatment effective dose is meant is enough to cause KSPi resistance in the cell to improve and/or strengthen the amount of the Growth Inhibition compound of KSP inhibitor.
5.5.1. effective dose
Can in cell culture or laboratory animal, determine the toxicity and the curative effect of described compound by standard pharmaceutical procedures, as determining LD
50(to 50% lethal dosage of colony) and ED
50(colony 50% in the treatment effective dosage).The dosage ratio of toxicity and curative effect is a therapeutic index, and it is expressed as ratio LD
50/ ED
50The compound that the preferred therapeutic index is big.Although can use the compound that shows toxic side effects, must careful design with the send delivery system of described compound target due to the site of affected tissue so that may be minimum, thereby reduce side effect to unaffected cells injury.
Can be used to customize the dosage that is used for human certain limit from the data that cell cultures is measured and zooscopy obtains.The dosage of described compound preferably drops on and comprises having seldom or do not have toxic ED
50The circulation composition scope.Dosage can change in this scope, depends on the formulation of use and the route of administration of employing.For any compound that is used for the inventive method, can measure from cell cultures at first and estimate the treatment effective dose.Can in animal model, customize dosage, to realize being included in the circulating plasma concentration range of the IC50 (that is, realizing the test compounds concentration of maximum half that suppresses of symptom) that determines in the cell culture.Described information can be used for useful dosage among more accurate definite mankind.For example, can be by the level in the high-efficient liquid phase color spectrometry blood plasma.
5.5.2. preparation and purposes
Can carry out preparationization with one or more physiology acceptable carriers or excipient to pharmaceutical composition used according to the invention according to conventional methods.
Therefore, can carry out preparationization, use or by oral, cheek or rectal administration for use in sucking or be blown into (through port or nose) to compound and the acceptable salt of physiology thereof and solvate.
For Orally administered, the form that pharmaceutical composition can adopt for example is, by ordinary method, with the acceptable excipient of pharmacy, as tackiness agent (as W-Gum, polyvinylpyrrolidone or the Vltra tears of pre-gelledization); Weighting agent (as lactose, Microcrystalline Cellulose or secondary calcium phosphate); Lubricant (as Magnesium Stearate, talcum or silicon-dioxide); Disintegrating agent (as yam starch or Vivastar P 5000); Or the tablet or the capsule of wetting agent (as sodium lauryl sulphate) preparation.Can be with method peridium patch well known in the art agent.Being used for Orally administered liquid preparation can be following form, for example, solution, syrup or suspension, or they can be used as the dryed product existence, water or other suitable excipient preparation before use.Can pass through ordinary method, adopt the pharmacy acceptable additive to prepare described liquid preparation, described additive such as suspension agent (as sorbitol syrup, derivatived cellulose or hydrogenation edible fat); Emulsifying agent (as Yelkin TTS or Sudan Gum-arabic); Non-water excipient (as the vegetables oil of Prunus amygdalus oil, grease, ethanol or fractional separation); And sanitas (as methyl p-hydroxybenzoate or propyl ester or Sorbic Acid).Described preparation also can contain suitable buffering salt, seasonings, tinting material and sweeting agent.
Be used for suitably preparationization of Orally administered preparation, to obtain the controlled release of active compound.
For using through cheek, composition can be taked the tablet or the lozenge form of preparationization in a conventional manner.
Use for suction, compound used according to the invention is routinely with the aerosol spray form, send by pressurized package or atomizer and to pass, wherein use suitable propelling agent, as Refrigerant 12, trichlorofluoromethane, dichloro tetrafluoro ethane, carbonic acid gas or other suitable gas.Under the situation of pressurized aerosol, can determine dose unit by valve is provided, to send the amount of passing metering.Can prepare and contain compound and suitable powder base, as the capsule and the cartridge case of for example gelatin that is used for sucker or insufflator of the powdered mixture of lactose or starch.
Can carry out preparationization to compound, be used for, for example pass through bolus injection or continuous infusion and parenteral administration by injection.The preparation that is used to inject may reside in unit dosage, for example, in ampoule or multi-agent container, wherein adds sanitas.Composition can be such as the suspension in oiliness or the aqueous carrier, solution or form of emulsion, and can contain preparation reagent, as suspension agent, stablizer and/or dispersion agent.Perhaps, activeconstituents can be a powder type, is used for using before use suitable carriers, as aseptic pyrogen-free water preparation.
Also can be in rectal compositions, as in suppository or the enema,retention compound carry out preparationization, described suppository or enema,retention for example can contain conventional suppository base, as cocoa ester or other glyceryl ester.
Except previously described preparation, also compound formulation can be turned to the storage preparation.Described prolonged action preparation can be by implanting (for example subcutaneous or intramuscular) or using by intramuscularly.Therefore, for example, can be with suitable polymerization or hydrophobic material (for example as the milk sap that can accept in the oil) or ion exchange resin, or as the slightly soluble derivative, for example as slightly soluble salt pair compound formulationization.
If desired, described composition may reside in packing or the distribution device, and described device can comprise the one or more unit dosage that contain activeconstituents.Packing can for example comprise metal or plastic foil, for example blister package.Can be in packing or the distribution device with using explanation.
5.5.3. route of administration
Suitable route of administration can comprise, for example oral, rectum, through mucous membrane, use through skin or enteron aisle; Stomach is sent outside and is passed, and comprises intramuscular, subcutaneous, intramedullary injection, and in the sheath, directly in the ventricle, in the intravenously, intraperitoneal, nose or intraocular injection.
Perhaps, can use, for example, compound is injected directly into affected area, usually to store or extended release preparation at the topical application compound rather than with system mode.
In addition, can be in the fixed drug delivery system of target, for example, at bag by drug administration in the liposome of specific antibody of influenced cell.The liposome target is scheduled cell, and absorbed by cell selective.
5.5.4. packing
If desired, composition may reside in the packing or distribution device that can comprise the one or more unit dosage that contain activeconstituents.Packing can for example comprise metal or plastic foil, for example blister package.Can be in packing or the distribution device with using explanation.Also can prepare and comprise the composition that comprise The compounds of this invention of preparationization in compatible pharmaceutical carriers, be placed in the suitable containers, and stick the label that is used for the treatment of the indication of pointing out.The appropriate conditions of pointing out on the label can comprise the treatment disease, as be characterised in that unusual or excessive STK6 or DNA destroy that response gene is expressed or active those.
6. embodiment
Following examples are for the present invention being described, being not intended to limit by any way the present invention.
6.1. embodiment 1:STK6 and TPX2 and KSP interact
This embodiment has illustrated in the screening siRNA library and the interactional gene of KSP gene inhibition agent.CIN8 is the Saccharomyces cerevisiae homologue of KSP.The deletion mutant of CIN8 can be survived, and has identified (CIN8 exists next really not so) many genes necessary under the non-existent condition of CIN8 (Geiser et al., 1997, Mol Biol Cell.8:1035-1050).Similarly, the destruction of inferring human homology's thing of these genes may not have more destructiveness to growth of tumour cell under the existence condition than it in the presence of the KSPi of suboptimal concentrations.Screening contains adjusting KSP inhibitor (1S)-the 1-{[(2S)-4-(2 in the siRNA library that it is reported with the siRNA of the homologue of CIN8 collaborative lethal 11 kinds of gene: CDC20, ROCK2, TTK, FZR1, BUB1, BUB3, BUB1B, MAD1L1, MAD2L1, DNCH1 and STK6, the 5-difluorophenyl)-and 2-phenyl-2,5-dihydro-1H-pyrroles-1-yl] carbonyl }-2-methyl propylamine (EC
50The gene of effect about 80nM).The sequence of the siRNA of fixed these the 11 kinds of genes of target is listed in Table I.Exist or do not exist<(1S)-1-{[(2S)-4-(2, the 5-the difluorophenyl)-2-phenyl-2 of EC10 concentration (25nM), 5-dihydro-1H-pyrroles-1-yl] carbonyl-condition of 2-methyl propylamine under with these siRNA transfections in the HeLa cell.Table I has also been listed the sequence that the difference target is decided the siRNA of luciferase, PTEN and KSP.
Be performed as follows the siRNA transfection: in transfection the day before yesterday, the cell that 100 microlitres are selected, as at DMEM/10% foetal calf serum (Invitrogen, Carlsbad, CA) grow to the about 90% cervical cancer HeLa cell (ATCC that converges in, Cat.No.CCL-2) with 1500 cells/well be planted in the 96 hole tissue culturing plates (Corning, Coming, NY).For each transfection, (Dharmacon Denver) mixes with siRNA from 5 microlitre serial dilutions of 20 micromole's liquid storages with 85 microlitre OptiMEM (Invitrogen).For each transfection, 5 microlitre OptiMEM are mixed with 5 microlitre Oligofectamine reagent (Invitrogen), and incubation 5 minutes at room temperature.Then 10 microlitre OptiMEM/Oligofectamine mixtures and OptiMEM/siRNA mixture are assigned in each test tube, mix, and at room temperature incubation 15-20 minute.10 microlitre transfection mixtures are distributed in each hole of 96 orifice plates, and under the condition of 37C and 5%CO2 incubation 4 hours.
After 4 hours, what add 100 microlitres/hole contains or does not contain 25nM (1S)-1-{[(2S)-4-(2, the 5-difluorophenyl)-and 2-phenyl-2,5-dihydro-1H-pyrroles-1-yl] carbonyl }-the DMEM/10% foetal calf serum of 2-methyl propylamine, with plate incubation 68 hours again under the condition of 37 ℃ and 5%CO2.Measure cell growth (referring to 5.2 joints) with Alamar Blue assay method.AlamarBlue measures and measures cellular respiration, and with observed value measuring as viable cell number.The interior environment of proliferative cell has higher reductibility than the interior environment of non-proliferative cell.Particularly, in breeding, NADPH/NADP, FADH/FAD, the ratio of FMNH/FMN and NADH/NAF increases.These metabolic intermediates can reduce alamarBlue, therefore, can be used to monitor cell proliferation.In the present embodiment, measure according to hereinafter carrying out alamarBlue, to determine 25nM KSP inhibitor (1S)-1-{[(2S)-4-(2, the 5-difluorophenyl)-2-phenyl-2,5-dihydro-1H-pyrroles-1-yl] carbonyl }-whether the existence of 2-methyl propylamine changed STK6siRNA transfection titration curve: after the transfection 72 hours, from the hole, remove substratum, and with containing 10% (vol/vol) alamarBlue reagent (Biosource InternationalInc., Camarillo, CA) and the DMEM/10% foetal calf serum (Invitrogen) of the 1M Hepes damping fluid tissue culture reagent (Invitrogen) of 0.001 volume replace.With plate 37 ℃ of incubations 2 hours, adopt then Softmax Pro 3.1.2 software (MolecularDevices) SpectraMax plus read the plate device (Molecular Devices, Sunnyvale, CA) on, 570 and the 600nm wavelength readings.The % reduction that contains the hole of sample is determined according to formula 1.Deduct the not % reduction in celliferous hole from the % reduction in the hole of containing sample, to determine to be higher than the % reduction of background.To there be or do not exist 25nM (1S)-1-{[(2S)-4-(2, the 5-difluorophenyl)-and 2-phenyl-2,5-dihydro-1H-pyrroles-1-yl] carbonyl }-compare with the reduction per-cent in the hole of the STK6siRNA transfection of certain titre and the hole of the siRNA transfection that target is decided luciferase under the condition of 2-methyl propylamine.Do not have (1S)-1-{[(2S)-4-(2, the 5-difluorophenyl)-2-phenyl-2,5-dihydro-1H-pyrroles-1-yl] carbonyl }-the % reduction numerical value that the hole of 0nM luciferase siRNA transfection calculated during 2-methyl propylamine is considered to 100%.
At (1S)-1-{[(2S)-4-(2, the 5-difluorophenyl)-2-phenyl-2,5-dihydro-1H-pyrroles-1-yl] carbonyl }-existence of 2-methyl propylamine is following, and three kinds of siRNA that target is decided STK6 (STK6-1, STK6-2 and STK6-3) show the inhibition growth of tumour cell.In the three, STK6-1 shows the strongest growth inhibitory activity in initial screening.In order to study this growth inhibitory activity is because hitting or missing the target activity of siRNA uses target decide three kinds of extra siRNA of STK6, and assesses all 6 kinds of siRNA and induce STK6 silence and growth inhibiting ability.Between reticent level of STK6 and growth-inhibiting, there is good dependency (Fig. 1).This dependency shows that growth-inhibiting is because the activity that hits (that is, STK6 destroys).Then at (1S)-1-{[(2S)-4-(2, the 5-difluorophenyl)-2-phenyl-2,5-dihydro-1H-pyrroles-1-yl] carbonyl-2-methyl propylamine exist or non-existent condition under, the contrast siRNA (negative control) of titration STK6-1 and luciferase is (Fig. 2).The interpolation of KSPi has been moved to the left about 5-10 doubly with the STK6-1 dose response curve.This concentration of KSPi does not have to strengthen the effect of the cell growth that is caused by luciferase siRNA.On the contrary, the dose response curve of target being decided the siRNA of PTEN is not moved by KSPi, and described PTEN has similar effect to the STK-6 cell growth.The siRNA of other STK6 also strengthens the effect of KSPi cell growth.Thus, the destruction of KSP has strengthened the effect of STK6siRNA cell growth.The research of the combination of the siRNA by adopting STK6 and KSP has obtained the further support to this, and described combination table reveals than arbitrary independent stronger growth inhibitory activity of siRNA.Do not influence cell growth owing to be used for KSPi concentration self of these experiments, active effect shows as collaborative, rather than synergetic KSPi to STK6siRNA.
Interaction consistent with the interactional evidence of the physiology of these genes in Africa xenopus (Giet et al., 1999, J Biol Chem.274:15005-5013) between human STK6 and the KSP.Particularly, the Africa xenopus homologue co of STK6 and KSP is at the mitotic spindle utmost point, and by immunoprecipitation, albumen shows molecular association.In addition, KSP is the substrate of STK6.
The growth-inhibiting of STK6siRNA shows that this gene pairs growth of tumour cell is necessary, and supports that STK6 is the research of anti-tumor target.Show that the collaborative interactional data that cause death between STK6 inhibitor and the KSPi show, carry out combination therapy with these compounds, may be than treating more effective with any independent compound.STK6 is overexpression in human tumor usually, comprise prognosis mala mammary cancer (van ' t Veer et al., 2002, Nature.2002415:530-536).The amplification of STK6 relevant with resistance (Anand et al., 2003, Cancer Cell.3:51-62) to taxol.Because KSPi and the identical target (mitotic spindle) of taxol influence, the overexpression of STK6 can reduce the validity of KSPi equally.This possibility is consistent with results of interaction between the inhibitor that shows KSPi and STK6, and should study in the clinical development process of KSPi.For example, KSPi may not have best effect in the patient with breast cancer of prognosis mala, because these tumours are tended to overexpression STK6.
Figure 17 shows the The selection result to the gene of KSPi sensitivity.The result shows that TPX2 also interacts with KSP.Be used to make the siRNA sequence of TPX2 silence also to list in Table I.
The tabulation of Table I siRNA
STK6-1 | GCACAAAAGCUUGUCUCCATT(SEQ?ID?NO:1) |
STK6-2 | UUGCAGAUUUUGGGUGGUCTT(SEQ?ID?NO:2) |
STK6-3 | ACAGUCUUAGGAAUCGUGCTT(SEQ?ID?NO:3) |
STK6-4 | CCUCCCUAUUCAGAAAGCUTT(SEQ?ID?NO:4) |
STK6-5 | GACUUUGAAAUUGGUCGCCTT(SEQ?ID?NO:5) |
STK6-6 | CACCCAAAAGAGCAAGCAGTT(SEQ?ID?NO:6) |
ROCK2-1 | AACCAGUCUAUUAGACGGCTT(SEQ?ID?NO:7) |
ROCK2-2 | GUGACUCUCCAUCUUGUAGTT(SEQ?ID?NO:8) |
ROCK2-3 | GUGGCCUCAAAGGCACUUATT(SEQ?ID?NO:9) |
CDC20-1 | CCCAUCACCUCAGUUGUUUTT(SEQ?ID?NO:10) |
CDC20-2 | GACCUGCCGUUACAUUCCUTT(SEQ?ID?NO:11) |
CDC20-3 | GGAGAACCAGUCUGAAAACTT(SEQ?ID?NO:12) |
TTK-1 | AUGCUGGAAAUUGCCCUGCTT(SEQ?ID?NO:13) |
TTK-2 | ACAACCCAGAGGACUGGUUTT(SEQ?ID?NO:14) |
TTK-3 | UAUGUUCUGGGCCAACUUGTT(SEQ?ID?NO:15) |
FZR1-1 | CCAGAUCCUUGUCUGGAAGTT(SEQ?ID?NO:16) |
FZR1-2 | CGACAACAAGCUGCUGGUCTT(SEQ?ID?NO:17) |
FZR1-3 | GAAGCUGUCCAUGUUGGAGTT(SEQ?ID?NO:18) |
BUB1-1 | CUGUAUGGGGUAUUCGCUGTT(SEQ?ID?NO:19) |
BUB1-2 | ACCCAUUUGCCAGCUCAAGTT(SEQ?ID?NO:20) |
BUB1-3 | CAGACUCCAUGUUUGCAGUTT(SEQ?ID?NO:21) |
BUB3-1 | UACAUUUGCCACAGGUGGUTT(SEQ?ID?NO:22) |
BUB3-2 | CAAUUCGUACUCCCCAAUGTT(SEQ?ID?NO:23) |
BUB3-3 | AGCUGCUUCAGACUGCUUCTT(SEQ?ID?NO:24) |
MAD1L1-1 | GACCUUUCCAGAUUCGUGGTT(SEQ?ID?NO:25) |
MAD1L1-2 | AGAGCAGAGCAGAUCCGUUTT(SEQ?ID?NO:26) |
MAD1L1-3 | CCAGCGGCUCAAGGAGGUUTT(SEQ?ID?NO:27) |
MAD2L2-1 | CCAUGACGUCGGACAUUUUTT(SEQ?ID?NO:28) |
MAD2L2-2 | GUGCUCUUAUCGCCUCUGUTT(SEQ?ID?NO:29) |
MAD2L2-3 | ACGCAAGAAGUACAACGUGTT(SEQ?ID?NO:30) |
DNCH1-1 | GCAAGUUGAGCUCUACCGCTT(SEQ?ID?NO:31) |
DNCH1-2 | UGGCCAGCGCUUACUGGAATT(SEQ?ID?NO:32) |
DNCH1-3 | GGCCAAGGAGGCGCUGGAATT(SEQ?ID?NO:33) |
BUB1B-1 | AUGACCCUCUGGAUGUUUGTT(SEQ?ID?NO:34) |
BUB1B-2 | UGCCAAUGAUGAGGCCACATT(SEQ?ID?NO:35) |
BUB1B-3 | GAAAGAACAGGUGAUCAGCTT(SEQ?ID?NO:36) |
Luciferase | CGUACGCGGAAUACUUCGATT(SEQ?ID?NO:37) |
KSP-1 | CUGGAUCGUAAGAAGGCAGTT(SEQ?ID?NO:38) |
KSP-2 | GGACAACUGCAGCUACUCUTT(SEQ?ID?NO:39) |
PTEN-1 | UGGAGGGGAAUGCUCAGAATT(SEQ?ID?NO:40) |
PTEN-2 | UAAAGAUGGCACUUUCCCGTT(SEQ?ID?NO:41) |
PTEN-3 | AAGGCAGCUAAAGGAAGUGTT(SEQ?ID?NO:42) |
TPX2 | UACUUGAAGGUGGGCCCAUTT(SEQ?ID?NO:1237) |
TPX2 | GAAAUCAGUUGCUGAGGGCTT(SEQ?ID?NO:1238) |
TPX2 | ACCUAGGACCGUCUUGCUUTT(SEQ?ID?NO:1239) |
6.2 embodiment 2: work in coordination with the screening that causes death with shRNA and siRNA
The present embodiment explanation makes CHEK1 and TP53 carry out the silence of RNAi mediation simultaneously, has caused the collaborative lethal effect among the human tumor cell.
Two problems have limited the potential of working in coordination with the screening that causes death with the RNAi method.At first, the proof of collaborative lethal effect need be hit and observe definite gene disruption inductive phenotype that causes death in the cell of genetically deficient or sudden change tending to first, does not then observe in only containing wild-type allele or proteic cell.Therefore, for the highly experiment of contrast, need be with hitting the clone of isogenic coupling the gene disruption to measuring collaborative lethal effect except first.For most of obtainable tumor cell lines, the clone of described coupling is to obtaining.Secondly, be subjected to the target obstruction of the siRNA of the different mRNA observations of competing each other calmly, effectively reduced the effectiveness of one or both siRNA of use by the trial of using dual siRNA transfection in cell, to produce two gene disruptions.Show in the present embodiment, can hit and send the excess revolutions of passing to dye in the liptinite of shRNA of gene, realize dual RNAi screening by use destroying first with the siRNA of fixed second gene of target.This method provides coupling (isogenic) clone to (shRNA adds deduct), and does not cause the competition between shRNA and the siRNA.In the present embodiment, set up cloned cell line, wherein the primary gene target is by the stably express silence of short hairpin RNA (shRNA).With the siRNA of fixed other gene of target these clones' transient transfection (excess revolutions is dyed) is not had obviously to influence the primary target silence that shRNA causes, shRNA does not influence the target silence that siRNA causes yet.Proved that with this method there is the collaborative lethal effect between following TP53 (p53) and the checkpoint kinase CHEK1 in lower concentration DNA disrupting agent Zorubicin.
Can by transient transfection send pass synthetic double-chain small disturbance RNA (siRNA) or from instantaneous importing or stable integration to genome recombinant vectors at cell inner expression short hairpin RNA (shRNA), thereby realization RNA interference (referring to, Paddison et al. for example, 2002, Genes Dev 16:948-958; Sui et al., 2002, Proc Natl Acad Sci USA 99:5515-5520; Yu et al., 2002, Proc Natl Acad Sci U S A 99:6047-6052; Miyagishi et al., 2002, Nat Biotechnol 20:497-500; Paul et al., 2002, Nat Biotechnol 20:505-508; Kwak et al., 2003, J Phannacol Sci 93:214-217; Brummelkamp et al., 2002, Science 296:550-553; Bodenetal., 2003, Nucleic Acids Res 31:5033-5038; Kawasaki et al., 2003, Nucleic Acids Res 31:700-707).Use coding tetracycline resistance markers and driving pRETRO-SUPER (pRS) carrier from H1 (RNA Pol III) promoter expression shRNA.The pRS-TP531026shRNA plasmid is from NKI library plasmid set deconvolution, and this is by gathering transform bacteria with this, and seeks the clone who only contains interested plasmid.Employed sequence is as follows: pRS-p53 1,026 19 aggressiveness sequence: GACTCCAGTGGTAATCTAC (SEQ ID NO:43); The primer of sequence-specific PCR: forward: GTAGATTACCACTGGAGTC (SEQ ID NO:44), oppositely: CCCTTGAACCTCCTCGTTCGACC (SEQ ID NO:45).Identify plasmid by sequence-specific PCR, and confirm by order-checking.By with FuGENE 6 (Roche) the transfection HCT116 cell that has pRS-TP53 1026 plasmids, prepare stable p 53 clones.After 48 hours cell is assigned in the 10cm culture dish that is added with the 1ug/ml tetracycline, keeps up to colony obvious (5-7 days).The clone is selected 96 orifice plates, in the 1ug/ml tetracycline, keep, and use TP53 and hGUS Pre-Developed to measure reagent (AppliedBiosystems), knock out by the TaqMan check.In order to measure instantaneous knocking out,, after 24 hours, gather in the crops RNA with Lipofectamine 2000 (Invitrogen) transfection HCT116 cell by pRS-TP53 1026 plasmids.By TaqMan assessment TP53 level.
Derive from colon tumor cell and be a plurality of tetracycline resistance TP53shRNA clones' (pRS-p53) of HCT116 the target silence (50%-96%) of analysis revealed different levels.Fig. 3 shows the TP53 expression level among clone A5 and the A11, and described clone shows the silence of highest level.TP53 silence among these clones has surpassed by transient transfection pRS-p53 to be sent and has been delivered to 24 hours observed silences (Fig. 3) behind the HCT116 cell.Transfection efficiency may the validity of restricted T P53shRNA in transient transfection.Perhaps, the cell with the reticent level of higher TP53 has obtained growth vigor in the clonal growth process.Adopt target to decide the shRNA (pRS-STK6:pRS-STK6 2,178 19 aggressiveness sequence: CATTGGAGTCATAGCATGT (SEQ ID NO:46)) of STK6, also observed the silence of the certain limit in the stable clone.But, these clones do not reach with TP53 clone in the silence of observed the same high level, its silence also is no more than the silence that obtains in instantaneous measurement.This may represent the selection of anti-high-level STK6 silence, because STK6 is the necessary gene of the growth of tumour cell in the culture.
In order to check the TP53 silence among the HCT116 clone whether to compete, dye this clone's cell with the set excess revolutions of CHEK1 specific siRNA with siRNA.The CHK1 set contains following three kinds of siRNA:CUGAAGAAGCAGUCGCAGUTT (SEQ ID NO:99); AUCGAUUCUGCUCCUCUAGTT (SEQID NO:98); And UGCCUGAAAGAGACUUGUGTT (SEQ ID NO:100).Find the reticent activity of the siRNA that the competitive TP53 of reduction of this siRNA set target is fixed.Show siRNA with 10nM or 100nM Oligofectamine (Invitrogen) transfection.For the CHK1 set,, send altogether and pass 100nM with three kinds of siRNA of 33.3nM transfection simultaneously.24 hours results RNA after transfection, and use CHK1 or TP53 and hGUS Pre-Developed to measure reagent (AppliedBiosystems), knock out by the TaqMan analysis and evaluation.Shown in Fig. 4 A, shRNA and siRNA gather the not silence of every kind of other target of competitive inhibition.Measure the inhibition that the known competitive siRNA by the shRNA of the siRNA of the transient transfection of identical sequence or stably express causes then.Shown in Fig. 4 B, three kinds of independent targets are decided KNSL1 (KNSLI210:GACCUGUGCCUUUUAGAGATT (SEQ ID NO:47); KNSLI211:GACUUCAUUGACAGUGGCCTT (SEQ ID NO:48); KNSLI212:AAAGGACAACUGCAGCUACTT (SEQ ID N0:49)) siRNA competitive inhibition is decided the silence (post on the left side) that the siRNA of the cotransfection of STK6 reaches by target.On the contrary, the influence that the silence that homology STK6shRNA causes in the clone of stable transfection is not dyed by the excess revolutions of KNSL1siRNA is even when adding competitor siRNA with high 10 times concentration also be so the post of (middle and the right).These experiments show, almost not competition between the siRNA of the shRNA of stably express and transient transfection.Observations during two kinds of different siRNA of that this competes each other with transfection together, fixed different mRNA of target is opposite, wherein effectively reduces the efficient of one or both siRNA of use.With the siRNA set transient transfection pRS and the pRS-p53HCT116 cell (embodiment 3 vide infra) of about 800 kinds of genes, and the effect of measuring cell growth by Alamar Blue assay method.Observed reaction much at one, do not shown reticent competitive inhibition the set of about 800 kinds of siRNA described in pRS cell and the pRS-p53 cell.
Subsequently, the excess revolutions that assessment CHEK1siRNA is integrated in the cell of stably express TP53shRNA is dyed, to determine whether to use it for the genetic interaction (SL) between these molecules of research.Inferred this interaction in the past, but, hindered its definite proof because shortage has enough specific reagent or gene knockout to the deduction effect of missing the target.By select to contain the clone that the A549 lung cancer cell line that free pRS carrier or pRS-p53 are arranged prepares coupling+/-TP53 expresses.A kind of cell in back shows>90% TP53mRNA silence.Then with contrast luciferase siRNA (luc, 100nM) or CHEK1siRNA set (100nM altogether; Each 33nM of 3 kinds of siRNA) two kinds of clones are dyed in excess revolutions, are exposing or be not exposed to DNA disrupting agent Zorubicin (Dox, their cell cycle spectrum of check under situation Fig. 5).Under the situation that lacks Dox, the cell cycle of pRS-p53 cell spectrum obviously is not different from the cell cycle spectrum of pRS cell.The transient transfection of CHEK1siRNA does not have influence not exist the cell cycle under the Dox situation to compose yet.But under the situation that Dox exists, as desired to the cell of expressing functional TP53, the pRS cells transfected has shown G1 and G2/M stagnates.The excess revolutions of CHEK1siRNA is dyed and has been caused crossing the G2 outpost of the tax office, and increases at the cell number of G1 blocking-up.Because cell keeps the TP53 function, they were stagnated and are not got back to the S phase in the G1 phase.
On the contrary, the pRS-p53 cell has lost the ability of stagnating at G1, and principal reaction handles and stagnate in the G2 phase in Dox, and this is consistent in the effect at the G1 outpost of the tax office with TP53.Cell cycle spectrum by the existing pRS-p53 cell of lucsiRNA excess revolutions hair dyeing does not change (Fig. 5).LucsiRNA even the part that does not cause TP53 to react are recovered (and the corresponding increase at G1 peak), show that this siRNA does not almost have to produce the competitive inhibition of and phenotype reticent to TP53.Therefore, estimate not exist CHEK1siRNA to gather the competitive inhibition of the TP53 silence that causes.In fact, react on Dox and handle, revealed remarkable change with the pRS-p53 cell of CHEK1 transient transfection at their cell cycle stave, the cell proportion in the S phase significantly increases, and has the DNA that inferior G1 (dead cell) measures.HCT116 cell kind at pRS and pRS-p53 stable transfection has also been observed similar discovery.Therefore, destroying in the time of by the G2 outpost of the tax office of the G1 outpost of the tax office of TP53 mediation and CHEK1 mediation, is lethal to the TP53-tumour cell, but is not lethal to the TP53+ tumour cell.
The siRNA of transfection is the silence that causes of the shRNA of competitive inhibition stably express not, and this discovery is unexpected.At present not clear why competitive intersection of siRNA suppresses reticent, and shRNA and siRNA are then really not so.This may point out, and this RNA of two types different step on biological chemistry enters the RNAi approach.
Figure 15 A-C shows the result of the reticent pair cell of CHEK1 to the influence of DNA destructive susceptibility.It is responsive that 15A:CHEK1 silence/inhibition makes the HeLa cell destroy DNA.It is responsive that 15B:CHEK1 silence/inhibition makes the p53-A549 cell destroy DNA.The 15C:CHEK1 silence does not make the HREP cell to the Zorubicin sensitivity.
6.3. embodiment 3: the gene that strengthens or weaken the cell killing that the DNA disrupting agent causes
Present embodiment has illustrated a kind of semi-automatic siRNA screening that is used to identify the gene that strengthens or weaken the cell killing that the DNA disrupting agent causes.Semi-automatic platform makes it possible to that ((loss-of-function) RNAi that siRNA ' s) carries out loss of function screens with siRNA.The toughener that the library of the siRNA of the about surely 800 kinds of Human genomes of target is used for identification of dna disrupting agent Zorubicin (Dox), camptothecine (Campto) and cis-platinum (Cis).In each screening, identified many genes (" hitting thing "), its destruction makes the cell killing sensitivity of cell to chemotherapeutics.(referring to Table III A-C).These some (as WEE1) promptings of hitting in the thing strengthen the active new target of chemotherapeutics commonly used; Other hits thing, and (BRCA1, BRCA2) prompting is used for the new therapy that the cancer that the sudden change of these genes causes is determined in heredity.
In order in the human cell, to carry out genescreen, set up the library of siRNA duplex.The about surely 800 kinds of genes of a kind of form target in this library, every kind of gene adopts three kinds of siRNA.This library extends to about surely 2, the 000 kinds of genes of target, extends further to fixed>7, the 000 kind of gene (2-3 kind siRNA/ gene) of target.This library comprises the siRNA of the fixed gene from " can be used as (druggable) genome of medicine " of target (using gene or the gene family of small molecules small molecules as medicine promptly).This library also comprises the siRNA of the fixed gene from " film group " (membranin) of target, to promote to identify the potential target of treatment antibody.Table III A-C has listed the sequence of the part of the siRNA that uses among this embodiment.In order to promote to carry out extensive siRNA screening, developed semi-automatic platform with this library.Before transfection, merge the isogenic three kinds of different siRNA of target phasing (total siRNA concentration is 100nM).Can by a people in less than 4 hours time with double complete library transfection in cell.Each siRNA set is checked 2-4 time in single experiment usually, and each experiment is repeated 2 times by Different Individual usually at least.Not on the same day or the screening undertaken by different people reached the repeatability that realizes.
The purpose of screening is to identify to make cell to cancer chemotherapeutic agent Dox, Campto commonly used and the target of Cis sensitivity.Dox (adriamycin) suppresses the activity of topoisomerase II (TopoII).TopoII mainly works in G2 and the M phase of cell cycle, and to untiing dna structure in case carry out chromosomal correct packing with separate very important.Campto suppresses topoisomerase I (TopoI).TopoI worked in the S phase, to discharge the twisting stress of advancing archaeal dna polymerase mixture.Campto is added cell in duplicating, cause replication fork to pause and the DNA splitting of chain.Cis causes dna adduct and chain crosslinked.Cis and Campto handle and cause replication fork to be stagnated and possible replication fork fracture, cause dsDNA fracture and necrocytosis.
The elementary screening of carrying out with each reagent is to carry out in the HeLa of TP53 defective cell.Gather transfection HeLa cell with siRNA, add medicine after 4 hours.Carry out the every kind drug concentrations of preliminary experiment to determine to use; Usually this is to cause the needed amount of 10%-20% growth-inhibiting (EC10 or EC20).Assessment in 72 hours after transfection+/-growth of the cell of medicine.
The structure of screening with Cis is shown among Fig. 6.Table II A shows the sensitization multiple that causes by the cis-platinum that on average surpasses the cis concentration of 400ng/ml and 500ng/ml.The figure shows under the situation that does not have medicine with the growth per-cent (log conversions) (X-axis) of siRNA set cells transfected and had growth per-cent (Y-axis) under the situation of medicine.Knocking out of some gene make to the pharmacological agent sensitization, its below diagonal lines, and some gene knock out the resistance of mediation to medicine, it is more than diagonal lines.The siRNA set that target is decided BRCA2 causes>10 times sensitization to Cis.The siRNA of BRCA1 set causes>3 times sensitization.The siRNA that target is decided kinases WEE1 and EPHB3 also cause to Cis>3 times sensitization.15 kinds of genes cause>2 times sensitization altogether.In similar screening, in each Dox and Campto screening, identified about 50 kinds cause to medicine>gene (referring to Table II b-C) of 2 times sensitization.Overlapping hereinafter between heterogeneic group discussed.
The design that is important to note that this screening is in order to find the toughener of pharmaceutical activity.Because the drug level cell growth of using produces considerably less effect, the co-inhibitor of medicine also cell growth produces considerably less effect.Like this, as expected, we have observed considerably less gene, and the destruction of described gene has been prevented pharmaceutical activity.A significant exception is the siRNA that target is decided polo sample kinases PLK, and its activity in the presence of Cis is lower.This may reflect that DNA destroys and PLK destroys the fact that causes cell cycle arrest.When cell cycle arrest had been induced in current a kind of treatment, the curative effect of a kind of treatment in back was lower.
Cause overlapping between the gene of different pharmaceutical sensitization in order to show, we compared the cell growth of different reagent-/+ratio (Fig. 7) of medicine (sensitization multiple).Relatively cause finding the kinase whose siRNA of some gene such as WEE1, make the kill and wound sensitivity of cell two kinds of reagent to the Dox sensitization with to the gene (Fig. 7, left side) of Cis sensitization.On the contrary, the siRNA that only decides breast cancer susceptibility gene BRCA2 with target has observed the strong sensitization (>10 times) that cell kills and wounds Cis.Relatively cause to the Campto sensitization with to the gene (Fig. 2, right side) of Cis sensitization, find the highest gene (BRCA2, BRCA1, EPHB3, WEE1 and ELK1) of scoring identical when treating with two kinds.
WEE1 destroys the observations prompting that causes all three kinds of reagent sensitizations, and this kinases is regulated the total DNA of all reagent is destroyed reaction.In the biological chemistry, human WEE1 avoids tenuigenin activated CDC2 kinases by the protection nucleus and destroys, and coordinates the transformation (Heald et al., 1993, Cell 74:463-474) between dna replication dna and the mitotic division.Other research prompting WEE1 repairs the composition at the outpost of the tax office at the DNA that the G2 phase of cell cycle works.Because most of human tumors are TP53 defectives, they lack the outpost of the tax office that the main TP53 that works in the G1 phase regulates, and therefore more depend on other outpost of the tax office (that is, they have normal pass card redundancy) than the healthy tissues of expressing TP53.Consider together to obtain data, show that WEE1 is provided for treating TP53 defective tumor treatment target, the survival of described tumour depends on G2 outpost of the tax office integrity.In fact, reported the radiosensitizer (that is, make cell to radiation inductive necrocytosis sensitivity) of the micromolecular inhibitor of WEE1, but the sensitization that this compound causes is moderate (Wang et al. as the TP53 deficient cells, 2001, Cancer Res.61:8211-7).Checked " hitting thing " ability that sensitized cell kills and wounds in other scope from these screenings in the function of the tumour cell outpost of the tax office: for example, by the cell of other tumor type and coupling+/-use other DNA disrupting agent in the TP53 function.
Consistent to the overlapping mechanism of action in the gene of Cis and Campto sensitization with these medicines.They all target decide the S phase, and the progress of replication fork is paused, cause the formation of dsRNA fracture material.On the contrary, Dox mainly works in the G2/M phase of cell cycle.Like this, may represent sensitization by BRCA1 and BRCA2 to the sensitization of Campto and Cis based on S phase specific mechanism.These results and the data consistent (D ' Andrea et al., 2003, Nat Rev Cancer3:23-34) that the research of the effect of BRCA1 and BRCA2 in the DNA destruction approach is obtained.In fact, now known these two kinds of genes all work in the DNA of the gene mediated relevant with congenital panhemocytopenia reparation approach; BRCA2 is identical with the FANCD1 of one of these genes.The cell that carries the defective in the BRCA approach has the susceptibility (Taniguchi et al., 2003, Nat Med.9:568-74) of increase to Cis.At present, the cancer patients with BRCA sudden change does not accept the treatment that target is decided their hereditary defect, but is making great efforts to detect platinum medicine (Couzin, 2003, Science 302:592) in these patients.
Consider together, these Notes of Key Datas siRNA Screening and Identification potential " reactor " colony (that is the tumour of BRCA pathway deficiency) of some DNA disrupting agent.Up to date, think that still only sub-fraction mammary gland and ovarian tumor are caused by the germ line mutation in the BRCA gene, because the tumour of distributing does not show the change in these genes usually.But nearest data show that other member's of BRCA approach inactivation of gene may the change than BRCA gene self more extensive (Marsit et al., 2004, Oncogene 23:1000-4) in the tumour of distributing.The siRNA in the bigger library of following use screens to have and helps identify other gene, and the destruction of described gene can be diagnosed the susceptibility to the DNA disrupting agent.In fact, in the siRNA library of expanding, represented a lot of known and predictions DNA-repair gene (as, comprise other congenital panhemocytopenia gene in the BRCA approach).The screening of appropriate designs also can be identified other molecule target useful to particular patient, has the BRCA pathway gene in the tumour of described particular patient and destroys.
Elementary screening is following to be carried out: screening contains the siRNA library of the siRNA of about 800 kinds of genes, the gene of adjusted cis-platinum (cis-diaminedichloroplatinum).Set (set of 3 kinds of siRNA of every kind of gene) screening library with the siRNA of 100nM (every kind of siRNA33nM).Exist or do not exist<condition of the cis-platinum of EC25 concentration (400ng/ml) under with these siRNA transfections in the HeLa cell.
In transfection the day before yesterday, the cell that 50 microlitres are selected, as at DMEM/10% foetal calf serum (Invitrogen, Carlsbad, CA) grow to the about 90% cervical cancer HeLa cell (ATCC that converges in, Cat.No.CCL-2) with 450 cells/well be planted in the 384 hole tissue culturing plates (Corning, Coming, NY).For each transfection, with 20 microlitre OptiMEM (Invitrogen) with (Dharmacon, Lafayette CO) mix from 2 microlitre siRNA of 10 micromole's liquid storages.For each transfection, 20 microlitre OptiMEM are mixed with 1 microlitre Oligofectamine reagent (Invitrogen), and incubation 5 minutes at room temperature.Then 20 microlitre OptiMEM/Oligofectamine mixtures and OptiMEM/siRNA mixture are assigned in each hole of 96 orifice plates, mix, and at room temperature incubation 15-20 minute.5 microlitre transfection mixtures are distributed in each hole of 384 orifice plates, and under the condition of 37 ℃ and 5%CO2 incubation 4 hours.4 difference 96 orifice plates that will contain different siRNA set are distributed on 384 orifice plates, and each accounts for 1/4th of 384 orifice plates.It all is to use the BioMek FX liquid processor with 96 hole distributor heads to carry out that all liquid shifts.
After 4 hours, add the DMEM/10% foetal calf serum that contains or do not contain the 2400ng/ml cis-platinum in 5 microlitres/hole, with plate incubation 68 hours under the condition of 37 ℃ and 5%CO2.Measure cell growth (referring to the 5.4.2.2 joint) with Alamar Blue assay method.AlamarBlue measures and measures cellular respiration, and with observed value measuring as viable cell number.The interior environment of proliferative cell has higher reductibility than the interior environment of non-proliferative cell.Particularly, in breeding, NADPH/NADP, FADH/FAD, the ratio of FMNH/FMN and NADH/NAF increases.These metabolic intermediates can reduce alamarBlue, therefore, can be used to monitor cell proliferation.After the transfection 72 hours, from the hole, remove substratum, and with containing 10% (vol/vol) alamarBlue reagent (Biosource International Inc., Camarillo, CA) and the DMEM/10% foetal calf serum (Invitrogen) of the 1M Hepes damping fluid tissue culture reagent (Invitrogen) of 0.001 volume replace.With plate 37 ℃ of incubations 2 hours, adopt Softmax Pro 3.1.2 software (Molecular Devices) at GeminiEM microtitration plate reader (Molecular Devices then, Sunnyvale, CA) on, excite with the 590nm emitted fluorescence by 545nm and to read plate.The relative fluorescence unit that gathers the hole of transfections from different siRNA deducts the not relative fluorescence unit in celliferous hole, to determine to be higher than the relative fluorescence unit of background level.The relative fluorescence unit in hole that exists or relative fluorescence unit and the target in the hole of siRNA set transfection decided the siRNA transfection of luciferase when not having cis-platinum is compared.Exist or when not having cis-platinum the relative fluorescence unit of luciferase siRNA cells transfected be considered to 100%.% growth with respect to luciferase by will not have medicine the time is plotted on X-axis, and the % growth with respect to luciferase when having medicine is plotted on Y-axis, has made comparison diagram.
Carried out secondary screens with HeLa cell, A549-pRS cell and A549-C7 cell.With the set (set of 3 kinds of siRNA of every kind of gene) of the siRNA of 100nM (every kind of siRNA 33nM), or with the single siRNA transfectional cell of 100nM.Under the condition that has or do not exist various concentration DNA disrupting agents with these siRNA transfections in the HeLa cell.The concentration of every kind of reagent is as follows: for the HeLa cell, and Zorubicin (10nM), camptothecine (6nM), cis-platinum (500ng/ml); For other clone, Zorubicin (200nM), camptothecine (200nM), cis-platinum (4ug/ml).
Following siRNA:WEE1 set, EPHB3 set, CHUK set, BRCA1 set, BRCA2 set and STK6 have been used.The sequence of the siRNA that uses is listed in Table III A.
Be performed as follows the siRNA transfection: in transfection the day before yesterday, the clone that 2000 microlitres are selected, as at DMEM/10% foetal calf serum (Invitrogen, Carlsbad, CA) grow to the about 90% cervical cancer HeLa cell (ATCC that converges in, Cat.No.CCL-2) be planted in the 6 hole tissue culturing plates with 45,000 cells/well.For each transfection, with 70 microlitre OptiMEM (Invitrogen) with (Dharmacon, Lafayette CO) mix from 5 microlitre siRNA of 20 micromole's liquid storages.For each transfection, 20 microlitre OptiMEM are mixed with 5 microlitre Oligofectamine reagent (Invitrogen), and incubation 5 minutes at room temperature.Then 25 microlitre OptiMEM/Oligofectamine mixtures are mixed with 75 microlitre OptiMEM/siRNA mixtures, and at room temperature incubation 15-20 minute.100 microlitre transfection mixtures are distributed in each hole of 6 orifice plates, and under the condition of 37 ℃ and 5%CO2 incubation 4 hours.
After 4 hours, the DMEM/10% foetal calf serum that contains or do not contain the DNA disrupting agent in 100 microlitres/hole is added each hole, to reach the final concentration of every kind of reagent mentioned above.With plate incubation 44 or 68 hours again under the condition of 37 ℃ and 5%CO2.Analyze cell cycle spectrum then from the sample of two time points (after the transfection 48 hours or 72 hours).
For cell cycle analysis, will be from the supernatant liquor and the cytomixis of gathering in the crops by tryptic digestion in each hole.Then with 1200rpm with centrifugal 5 minutes of mixture.With 70% ice-cold ethanol with about 30 minutes of cell fixation.With PBS once, be suspended in 0.5ml again and contain iodate third ingot (among the PBS of 10 micrograms/ml) and RNA enzyme A (1mg/ml), and 37 ℃ of following incubations 30 minutes with the fixed cell washing.(Becton Dickinson) carries out flow cytometry with the FACSCalibur flow cytometer, and with FlowJo software (TreeStar, Inc) analytical data.Measure necrocytosis with inferior G1 cell mass.If (siRNA+DMSO) summation of the inferior G1 cell mass of sample and (Luc+ medicine) sample thinks that greater than the inferior G1 cell mass of (siRNA+ medicine) sample the siRNA silence makes cell destroy sensitization to DNA.
Fig. 9-14 shows the result of secondary screens.Fig. 9 A-9C represents that the silence of WEE1 makes the HeLa cell destroy responsive to Dox, Campto and Cis inductive DNA.Fig. 9 D-9I shows that the silence of WEE1 makes the p53-A549 cell destroy sensitivity to Dox, Campto and Cis inductive DNA, but it is responsive that the p53+A549 cell is destroyed described DNA.Figure 10 A-10C shows that the silence of EPHB3 makes HeLa cell and p53-A549C7 destroy sensitivity to Dox, Campto and Cis inductive DNA, makes the p53+A549pRS cell lower to the degree of Dox, Campto and Cis inductive DNA destruction sensitivity.Figure 11 A-11C shows that the STK6 silence makes HeLa cell and p53-A549C7 destroy sensitivity to Dox, Campto and Cis inductive DNA, makes the p53+A549pRS cell lower to the degree of Dox, Campto and Cis inductive DNA destruction sensitivity.Figure 12 A-12C shows that the silence of BRCA1 makes HeLa cell and p53-A549C7 cell destroy responsive to Dox, Campto and Cis inductive DNA.It is responsive that the silence of BRCA also makes the p53+A549pRS cell destroy Cis inductive DNA with lower degree, but do not make the p53+A549pRS cell destroy responsive to Campto and Cis inductive DNA.Figure 13 A-13B shows that the silence of BRCA2 makes HeLa cell and p53-A549C7 cell destroy responsive to Dox, Campto and Cis inductive DNA.It is responsive that Figure 13 C shows that the silence of BRCA makes the p53+A549pRS cell destroy Cis inductive DNA with lower degree, but do not make the p53+A549pRS cell destroy responsive to Campto and Cis inductive DNA.Figure 14 A-14B shows that the silence of CHUK makes the HeLa cell destroy responsive to Dox, Campto and Cis inductive DNA.Figure 14 C shows that the silence of CHUK makes the p53-A549pRS cell destroy responsive to Campto and Cis inductive DNA.Figure 14 D shows that the silence of CHUK does not make the 53+A549pRS cell destroy responsive to Campto and Cis inductive DNA.
The average sensitization multiple that Table II A cis-platinum causes
Gene I | The gene title | Average sensitization multiple | Standard deviation | |
1 | 2514 | PLK | 0.302987553 | 0.122442 |
2 | 3099 | PLK | 0.344442634 | 0.157221 |
3 | 3433 | PLK | 0.415618617 | 0.142888 |
4 | 3266 | PLK | 0.471258534 | 0.273419 |
5 | 3006 | PLK | 0.573026377 | 0.295022 |
6 | 3534 | PLK | 0.580135373 | 0.403069 |
7 | 3806 | C10orf3 | 0.581678284 | 0.122098 |
8 | 3322 | CCNA2 | 0.603615299 | 0.027899 |
9 | 3805 | C20orf1 | 0.618083836 | 0.081029 |
10 | 3423 | NM_006101 | 0.640054878 | 0.131981 |
11 | 3464 | INSR | 0.67184541 | 0.043498 |
12 | 3722 | TLK2 | 0.680201667 | 0.164793 |
13 | 3731 | CSNK1E | 0.70971928 | 0.169767 |
14 | 3261 | ERBB2 | 0.721804997 | 0.095466 |
15 | 3093 | PIK3CG | 0.730517635 | 0.16341 |
16 | 3391 | PLK | 0.73566872 | 0.438713 |
17 | 3813 | ANLN | 0.742286686 | 0.076826 |
18 | 3687 | CAMK4 | 0.763785182 | 0.078326 |
19 | 3838 | PRKAA2 | 0.768128477 | 0.098461 |
20 | 2702 | 2702 | 0.77422078 | 0.032982 |
21 | 3435 | FLT3 | 0.786069641 | 0.033061 |
22 | 3740 | STK35 | 0.786251834 | 0.241352 |
23 | 3826 | NM_015694 | 0.78668619 | 0.158833 |
24 | 3113 | CNK | 0.789751097 | 0.074976 |
25 | 3648 | CLK1 | 0.795962486 | 0.119858 |
26 | 3397 | BUB3 | 0.798897309 | 0.041819 |
27 | 2982 | CDC2L2 | 0.803290264 | 0.28261 |
28 | 2975 | NEK4 | 0.804972926 | 0.092313 |
29 | 3003 | FER | 0.806761229 | 0.283308 |
30 | 3776 | NOTCH2 | 0.807626974 | 0.090463 |
31 | 3600 | RRM2B | 0.807791139 | 0.116058 |
32 | 3303 | CDKN2D | 0.808236038 | 0.106543 |
33 | 3536 | PIK3C3 | 0.811623871 | 0.072924 |
34 | 3491 | PRKCE | 0.818554314 | 0.081903 |
35 | 3181 | ST5 | 0.820227877 | 0.105561 |
36 | 3812 | CDCA8 | 0.825194175 | 0.149709 |
37 | 3525 | NOTCH4 | 0.826075824 | 0.135465 |
38 | 3182 | MYCN | 0.826997754 | 0.074996 |
39 | 2992 | PRKR | 0.83026411 | 0.107682 |
40 | 2972 | KSR | 0.840737073 | 0.220722 |
41 | 3359 | TUBA1 | 0.841656288 | 0.176344 |
42 | 3183 | NM_005200 | 0.843755002 | 0.126232 |
43 | 2961 | PIM1 | 0.846814316 | 0.1791 |
44 | 3814 | HMMR | 0.848584565 | 0.089675 |
45 | 3326 | CCT7 | 0.850805908 | 0.139648 |
46 | 3819 | TACC3 | 0.851051224 | 0.151449 |
47 | 3495 | FGFR2 | 0.851658058 | 0.169414 |
48 | 2952 | PRKG1 | 0.853083744 | 0.103483 |
49 | 3680 | CLK3 | 0.853111421 | 0.029348 |
50 | 3650 | NM_025195 | 0.855769333 | 0.097938 |
51 | 3635 | STAT1 | 0.856732819 | 0.045221 |
52 | 3487 | MAP2K3 | 0.858609643 | 0.046727 |
53 | 3831 | CLSPN | 0.865300447 | 0.043122 |
54 | 3416 | IKBKE | 0.868770694 | 0.033925 |
55 | 3693 | NEK9 | 0.871865115 | 0.272749 |
56 | 3686 | MAP3K8 | 0.872321606 | 0.276021 |
57 | 3677 | HCK | 0.874242862 | 0.099478 |
58 | 3509 | KIF21A | 0.876152348 | 0.070276 |
59 | 3666 | PAK6 | 0.877347139 | 0.070142 |
60 | 3563 | RAB3A | 0.877392452 | 0.07511 |
61 | 2993 | SRMS | 0.877914429 | 0.052743 |
62 | 3658 | STK18 | 0.884409716 | 0.022945 |
63 | 3153 | RB1 | 0.884802012 | 0.066909 |
64 | 3000 | BMX | 0.88790935 | 0.05788 |
65 | 3784 | MAPK8 | 0.888444434 | 0.124134 |
66 | 3503 | EGR1 | 0.8888158 | 0.172111 |
67 | 3578 | RREB1 | 0.889406356 | 0.126074 |
68 | 3085 | KIF5C | 0.889747874 | 0.062749 |
69 | 3431 | NM_018454 | 0.893082893 | 0.124062 |
70 | 2954 | ROCK2 | 0.893933798 | 0.055935 |
71 | 2922 | NM_004783 | 0.89487587 | 0.052019 |
72 | 3631 | WISP2 | 0.895799222 | 0.04132 |
73 | 3752 | CCNB3 | 0.895903064 | 0.014712 |
74 | 3808 | CKAP2 | 0.897429532 | 0.077036 |
75 | 3399 | HSPCB | 0.898588123 | 0.283379 |
76 | 3251 | ABL1 | 0.899747173 | 0.09061 |
77 | 3695 | PRKAA1 | 0.899926191 | 0.099839 |
78 | 3319 | CCND1 | 0.901342596 | 0.14162 |
79 | 3786 | FRAP1 | 0.901481586 | 0.064389 |
80 | 2964 | RIPK2 | 0.901658094 | 0.057156 |
81 | 3179 | PDGFB | 0.902358454 | 0.054703 |
82 | 2987 | RNASEL | 0.90485908 | 0.109916 |
83 | 3086 | KIF11 | 0.905925473 | 0.044166 |
84 | 3610 | LEF1 | 0.906026445 | 0.269465 |
85 | 3798 | ACTR2 | 0.9086166 | 0.162743 |
86 | 3088 | KIF13B | 0.912159346 | 0.09222 |
87 | 3332 | CDC5L | 0.912625936 | 0.141471 |
88 | 3711 | LIMK1 | 0.912891621 | 0.150911 |
89 | 3775 | NOTCH1 | 0.914649314 | 0.049686 |
90 | 3743 | RAGE | 0.915875434 | 0.062887 |
91 | 3410 | RPS27 | 0.916611322 | 0.14842 |
92 | 3403 | AURKC | 0.917162845 | 0.112884 |
93 | 3197 | ARHB | 0.917549671 | 0.07581 |
94 | 3145 | C20orf23 | 0.918517448 | 0.040236 |
95 | 2980 | RIPK1 | 0.918693241 | 0.035801 |
96 | 3646 | NM_005781 | 0.919629184 | 0.074213 |
97 | 3256 | CDC2L1 | 0.920311861 | 0.161437 |
98 | 3171 | VHL | 0.921197139 | 0.154964 |
99 | 3661 | FGR | 0.921903863 | 0.062718 |
100 | 2978 | AB067470 | 0.922713135 | 0.058126 |
101 | 2983 | GUCY2C | 0.922891001 | 0.132499 |
102 | 3557 | JUND | 0.923386231 | 0.212516 |
103 | 3573 | NM_016848 | 0.924255509 | 0.025747 |
104 | 3783 | KRAS2 | 0.924335869 | 0.031975 |
105 | 3833 | ATR | 0.925151796 | 0.036459 |
106 | 3762 | MCC | 0.926766797 | 0.063215 |
107 | 2934 | IRAK2 | 0.927137542 | 0.090048 |
108 | 3311 | CDK10 | 0.927487493 | 0.197303 |
109 | 3230 | MAP2K1 | 0.929528292 | 0.087866 |
110 | 3461 | KIT | 0.929864607 | 0.065105 |
111 | 3581 | RASGRP1 | 0.930046334 | 0.085936 |
112 | 3782 | SOS1 | 0.93078276 | 0.086957 |
113 | 3348 | DCK | 0.932934579 | 0.140927 |
114 | 3518 | NFKB1 | 0.933538042 | 0.254776 |
115 | 3692 | AB007941 | 0.934031479 | 0.122891 |
116 | 2936 | SGKL | 0.935268856 | 0.12869 |
117 | 3788 | PRKCE | 0.935825459 | 0.100437 |
118 | 3791 | NM_005200 | 0.937373151 | 0.124551 |
119 | 3827 | NM_018123 | 0.938752687 | 0.120885 |
120 | 3343 | CENPJ | 0.939276361 | 0.15064 |
121 | 3413 | KIF23 | 0.940719223 | 0.224476 |
122 | 3540 | PPP2CB | 0.940825549 | 0.07786 |
123 | 3559 | RAP1GDS1 | 0.941186098 | 0.092318 |
124 | 2943 | DYRK2 | 0.941751587 | 0.079768 |
125 | 3090 | KIF3C | 0.942994713 | 0.043187 |
126 | 3306 | CDC14A | 0.943159212 | 0.105314 |
127 | 3572 | RASA3 | 0.943756386 | 0.044924 |
128 | 3822 | GTSE1 | 0.944755556 | 0.2332 |
129 | 3351 | ESR1 | 0.944920378 | 0.153622 |
130 | 3258 | MOS | 0.9460337 | 0.090205 |
131 | 3601 | POLE | 0.94708241 | 0.126731 |
132 | 2960 | LYN | 0.947322877 | 0.19148 |
133 | 3828 | KIF20A | 0.950773558 | 0.183938 |
134 | 3778 | VHL | 0.951938861 | 0.232481 |
135 | 3196 | ARHI | 0.952842248 | 0.058681 |
136 | 3566 | JUN | 0.95294025 | 0.127285 |
137 | 3240 | MAPK12 | 0.953564717 | 0.071586 |
138 | 3184 | TSG101 | 0.954002138 | 0.04823 |
139 | 3714 | NM_013355 | 0.954197885 | 0.12488 |
140 | 3364 | HPRT1 | 0.954414394 | 0.271771 |
141 | 3685 | LTK | 0.954443302 | 0.285398 |
142 | 3751 | BCR | 0.954467451 | 0.121004 |
143 | 3434 | DDX6 | 0.954790843 | 0.082973 |
144 | 3298 | CCNE1 | 0.955113281 | 0.080149 |
145 | 3449 | TBK1 | 0.955301632 | 0.018969 |
146 | 3795 | NR4A2 | 0.955557277 | 0.096686 |
147 | 3739 | NM_017886 | 0.955581637 | 0.103771 |
148 | 3471 | MAPK10 | 0.956705519 | 0.068765 |
149 | 3139 | XM_095827 | 0.956993628 | 0.217327 |
150 | 3545 | IRS2 | 0.957861116 | 0.058638 |
151 | 2985 | MKNK1 | 0.958784274 | 0.02755 |
152 | 3618 | DVL2 | 0.958860428 | 0.145917 |
153 | 3726 | MAPKAPK2 | 0.95922853 | 0.071282 |
154 | 3678 | PFTK1 | 0.960709464 | 0.043435 |
155 | 3821 | ASPM | 0.961220945 | 0.129044 |
156 | 3163 | THRA | 0.96204376 | 0.138031 |
157 | 3101 | MAPK14 | 0.962194967 | 0.089772 |
158 | 3561 | FOS | 0.96220097 | 0.038394 |
159 | 3133 | XM_168069 | 0.962355545 | 0.075119 |
160 | 3443 | EPS8 | 0.962670509 | 0.080284 |
161 | 3117 | ATM | 0.963448158 | 0.17684 |
162 | 3401 | HDAC1 | 0.963594921 | 0.053087 |
163 | 3799 | ACTR3 | 0.96385153 | 0.106281 |
164 | 3733 | MYLK2 | 0.96390586 | 0.071956 |
165 | 3801 | PSEN1 | 0.96432309 | 0.133399 |
166 | 3716 | ULK1 | 0.964714374 | 0.172756 |
167 | 2977 | RIPK3 | 0.965321488 | 0.288006 |
168 | 3571 | VAV1 | 0.966085791 | 0.040696 |
169 | 2946 | NM_017719 | 0.966726854 | 0.070416 |
170 | 3459 | EGFR | 0.968475197 | 0.03989 |
171 | 3835 | CHEK2 | 0.968492479 | 0.077394 |
172 | 3125 | NM_031217 | 0.968705878 | 0.158815 |
173 | 3308 | CDKN2B | 0.970454697 | 0.030316 |
174 | 3458 | ARAF1 | 0.972126514 | 0.150383 |
175 | 3162 | MADH2 | 0.97228749 | 0.077251 |
176 | 2949 | MYO3B | 0.973636618 | 0.06916 |
177 | 3664 | STK17A | 0.975343312 | 0.06811 |
178 | 3488 | AURKB | 0.975742132 | 0.178321 |
179 | 3112 | KNSL7 | 0.976665103 | 0.157911 |
180 | 3485 | DHX8 | 0.978053596 | 0.073262 |
181 | 3809 | CDCA3 | 0.979002265 | 0.231181 |
182 | 3161 | WT1 | 0.979221693 | 0.114838 |
183 | 3513 | ROS1 | 0.979271577 | 0.121589 |
184 | 3185 | VCAM1 | 0.979438257 | 0.069759 |
185 | 3553 | CKS1B | 0.979465469 | 0.05555 |
186 | 3763 | NM_016231 | 0.980990574 | 0.123555 |
187 | 3245 | AXL | 0.981022783 | 0.078724 |
188 | 3334 | CUL4B | 0.981485893 | 0.048462 |
189 | 3193 | FGF3 | 0.981515057 | 0.075982 |
190 | 3335 | CDK5R2 | 0.983188137 | 0.095535 |
191 | 3455 | MAP2K4 | 0.984383299 | 0.095921 |
192 | 2925 | FYN | 0.984597535 | 0.177611 |
193 | 3215 | MAD2L1 | 0.984674289 | 0.166817 |
194 | 3519 | NTRK1 | 0.985625526 | 0.225903 |
195 | 2541 | 2541 | 0.985658529 | 0.02934 |
196 | 3109 | KIF1C | 0.985891536 | 0.162583 |
197 | 3792 | ARHGEF1 | 0.985986394 | 0.150503 |
198 | 3374 | POLR2A | 0.986875398 | 0.174675 |
199 | 3362 | NR3C1 | 0.98711375 | 0.09249 |
200 | 3231 | ILK | 0.987500124 | 0.068942 |
201 | 3166 | PMS1 | 0.987593476 | 0.040016 |
202 | 3703 | AK024504 | 0.988238947 | 0.078314 |
203 | 3707 | TXK | 0.98900485 | 0.138186 |
204 | 3323 | CDK5R1 | 0.989595604 | 0.176376 |
205 | 3180 | CD44 | 0.990121058 | 0.090413 |
206 | 3630 | WISP3 | 0.990225627 | 0.071631 |
207 | 3576 | GRAP | 0.990505346 | 0.120959 |
208 | 3800 | CHFR | 0.99369692 | 0.117429 |
209 | 3142 | KIF25 | 0.993932342 | 0.044087 |
210 | 3160 | TACSTD1 | 0.994447265 | 0.128513 |
211 | 3497 | EPHA8 | 0.99446771 | 0.015206 |
212 | 3757 | CLK4 | 0.995683284 | 0.166859 |
213 | 3645 | CASK | 0.996395727 | 0.07959 |
214 | 3357 | PRIM2A | 0.998092371 | 0.117247 |
215 | 3594 | RAP2A | 0.99814842 | 0.142818 |
216 | 3796 | ARHGEF6 | 0.998577367 | 0.091413 |
217 | 3767 | FZD3 | 0.99921132 | 0.096395 |
218 | 3715 | CDC42BPA | 0.999524848 | 0.196389 |
219 | 2938 | ALS2CR7 | 0.99966653 | 0.007718 |
220 | 3419 | RFC4 | 0.999756476 | 0.079342 |
221 | 63 | M15077 | 1 | 0 |
222 | 3672 | SYK | 1.000094306 | 0.028316 |
223 | 3832 | ATM | 1.00019002 | 0.091546 |
224 | 3627 | CTNNA1 | 1.000291459 | 0.215453 |
225 | 2984 | EPHB6 | 1.000603948 | 0.098044 |
226 | 3200 | REL | 1.000616585 | 0.104464 |
227 | 3492 | PRKCQ | 1.000785085 | 0.103181 |
228 | 3478 | EPHA2 | 1.001756995 | 0.101444 |
229 | 3539 | PLCG2 | 1.002008086 | 0.072305 |
230 | 3378 | NM_006009 | 1.002912861 | 0.019886 |
231 | 3381 | POLR2B | 1.003653073 | 0.021542 |
232 | 3452 | JAK1 | 1.00410017 | 0.246916 |
233 | 2926 | AF172264 | 1.0043601 | 0.195291 |
234 | 3641 | TYRO3 | 1.005062954 | 0.13144 |
235 | 3750 | CAMK2A | 1.005879519 | 0.197981 |
236 | 3595 | FEN1 | 1.006107713 | 0.1559 |
237 | 3375 | AHCY | 1.006847357 | 0.098914 |
238 | 3367 | DHFR | 1.007697409 | 0.048476 |
239 | 3555 | RASA1 | 1.007907357 | 0.060107 |
240 | 3246 | RPS6KB1 | 1.008295705 | 0.098199 |
241 | 3551 | STAT3 | 1.008697776 | 0.069559 |
242 | 3708 | RPS6KC1 | 1.008738004 | 0.158539 |
243 | 3820 | NM_018410 | 1.008803441 | 0.00423 |
244 | 3548 | RAC1 | 1.009000664 | 0.10905 |
245 | 3527 | DTX2 | 1.00940399 | 0.082767 |
246 | 3339 | CCNB2 | 1.009625325 | 0.321434 |
247 | 3226 | RBX1 | 1.01029159 | 0.235969 |
248 | 3473 | DAPK1 | 1.010335394 | 0.065266 |
249 | 3469 | AAK1 | 1.011653085 | 0.153819 |
250 | 3517 | MYC | 1.011855757 | 0.088032 |
251 | 3005 | MERTK | 1.011910266 | 0.139112 |
252 | 3294 | CCNF | 1.01355402 | 0.151217 |
253 | 3392 | BIRC5 | 1.014018575 | 0.147292 |
254 | 3533 | HES7 | 1.016868954 | 0.209244 |
255 | 3524 | NOTCH3 | 1.017285472 | 0.068877 |
256 | 3587 | VAV3 | 1.018129173 | 0.062737 |
257 | 3425 | DLG7 | 1.018264827 | 0.037325 |
258 | 3674 | CSNK1D | 1.018650699 | 0.087521 |
259 | 3380 | TUBG2 | 1.019248432 | 0.027697 |
260 | 3486 | RPS6KA3 | 1.019985527 | 0.050031 |
261 | 3746 | HUNK | 1.020779918 | 0.082372 |
262 | 3535 | SKP2 | 1.021142953 | 0.100064 |
263 | 3797 | ARHGEF9 | 1.021635562 | 0.137783 |
264 | 2969 | NM_014916 | 1.021887811 | 0.080467 |
265 | 3460 | CSK | 1.022085366 | 0.135805 |
266 | 3132 | KIF23 | 1.023806782 | 0.129496 |
267 | 2963 | MAP3K11 | 1.0242873 | 0.065223 |
268 | 3702 | MAP3K13 | 1.024294874 | 0.083014 |
269 | 3382 | TUBB | 1.025915608 | 0.049937 |
270 | 3237 | CDC7 | 1.025994603 | 0.096409 |
271 | 3592 | SOS2 | 1.026235513 | 0.178995 |
272 | 3365 | PRIM1 | 1.02653855 | 0.104798 |
273 | 3570 | RALGDS | 1.027460697 | 0.084873 |
274 | 3224 | FBXO5 | 1.029155584 | 0.154545 |
275 | 3585 | GAB1 | 1.029481526 | 0.06077 |
276 | 3414 | HDAC7A | 1.030424895 | 0.139587 |
277 | 3514 | HRAS | 1.030481671 | 0.09281 |
278 | 3597 | SHMT2 | 1.031207997 | 0.180827 |
279 | 3657 | PCTK1 | 1.031839128 | 0.067828 |
280 | 3257 | IGF1R | 1.03192729 | 0.10264 |
281 | 3773 | WNT2 | 1.032309731 | 0.174004 |
282 | 3625 | CTBP2 | 1.032538009 | 0.159078 |
283 | 3302 | CDK8 | 1.032760545 | 0.077771 |
284 | 3409 | TTK | 1.033113517 | 0.089383 |
285 | 3465 | EPHA1 | 1.033516487 | 0.127809 |
286 | 3705 | NM_012119 | 1.033751364 | 0.107948 |
287 | 2966 | NM_033266 | 1.035012335 | 0.097641 |
288 | 2999 | FES | 1.035558582 | 0.12725 |
289 | 3474 | CSNK2A1 | 1.036151218 | 0.085057 |
290 | 3824 | MAPRE3 | 1.036197838 | 0.092706 |
291 | 3094 | KIF3A | 1.036464942 | 0.119921 |
292 | 3769 | PLAU | 1.037390211 | 0.064893 |
293 | 3213 | NM_016238 | 1.037745909 | 0.138786 |
294 | 2950 | NEK6 | 1.038291854 | 0.149776 |
295 | 3815 | MAPRE2 | 1.038305947 | 0.217709 |
296 | 3543 | PDK2 | 1.038311221 | 0.197679 |
297 | 3437 | FGFR1 | 1.0383269 | 0.283643 |
298 | 3542 | PPP2CA | 1.039357671 | 0.194352 |
299 | 3511 | XM_168069 | 1.039370913 | 0.155262 |
300 | 3002 | CRKL | 1.039983971 | 0.101129 |
301 | 3398 | HDAC11 | 1.041663934 | 0.099406 |
302 | 3675 | ADRBK1 | 1.041741459 | 0.084419 |
303 | 3623 | CTNND1 | 1.042210238 | 0.105978 |
304 | 3268 | CDC25C | 1.042762357 | 0.02726 |
305 | 3633 | CTBP1 | 1.042818569 | 0.143171 |
306 | 3804 | NM_024322 | 1.042895573 | 0.05266 |
307 | 3526 | HES6 | 1.043146787 | 0.059244 |
308 | 2947 | NM_007064 | 1.043456689 | 0.080305 |
309 | 2979 | PAK2 | 1.043537793 | 0.115188 |
310 | 2959 | PIM2 | 1.043942064 | 0.050352 |
311 | 3602 | MCM3 | 1.044071108 | 0.23865 |
312 | 3665 | PAK4 | 1.044246523 | 0.052921 |
313 | 3421 | ORC6L | 1.044825423 | 0.241726 |
314 | 3745 | CAMKK2 | 1.044966032 | 0.032844 |
315 | 3736 | PTK7 | 1.045008777 | 0.118965 |
316 | 3119 | CDKN1B | 1.045154749 | 0.026803 |
317 | 3643 | DDR2 | 1.045796426 | 0.123748 |
318 | 3603 | POLS | 1.046796283 | 0.090212 |
319 | 3346 | CCNK | 1.047737442 | 0.148152 |
320 | 3438 | DTR | 1.04975054 | 0.139619 |
321 | 2942 | TTN | 1.050944386 | 0.134575 |
322 | 2937 | NM_025052 | 1.051593448 | 0.052118 |
323 | 3577 | RAB2L | 1.051977248 | 0.073992 |
324 | 3203 | ITGA5 | 1.052197011 | 0.109443 |
325 | 3599 | DTYMK | 1.052206896 | 0.147041 |
326 | 3373 | top2A | 1.053946926 | 0.061071 |
327 | 3222 | PTTG1 | 1.054934465 | 0.059734 |
328 | 3154 | MADH4 | 1.055367142 | 0.392285 |
329 | 3829 | KIF2C | 1.056017438 | 0.187684 |
330 | 3652 | PDGFRA | 1.056020056 | 0.084537 |
331 | 2944 | MARK1 | 1.056491568 | 0.161232 |
332 | 3656 | PRKCN | 1.056755878 | 0.177943 |
333 | 3626 | DVL3 | 1.058711269 | 0.19647 |
334 | 3802 | NOTCH3 | 1.059031918 | 0.117495 |
335 | 3127 | MAPK1 | 1.059441261 | 0.070449 |
336 | 3549 | PIK3R2 | 1.059495493 | 0.178697 |
337 | 2935 | MAPK6 | 1.060533709 | 0.075031 |
338 | 3307 | CDC6 | 1.060858236 | 0.093933 |
339 | 3260 | STK11 | 1.061848445 | 0.120762 |
340 | 3766 | S100A2 | 1.062832073 | 0.174576 |
341 | 3457 | BAD | 1.063944791 | 0.07637 |
342 | 3347 | top1 | 1.06614481 | 0.169748 |
343 | 3450 | MAP3K2 | 1.066638971 | 0.166869 |
344 | 3164 | MYCL1 | 1.066666964 | 0.198532 |
345 | 3412 | KIF25 | 1.068536113 | 0.202887 |
346 | 3317 | CCNI | 1.068966464 | 0.126188 |
347 | 3550 | PLCG1 | 1.069052894 | 0.123064 |
348 | 3668 | DAPK3 | 1.069120278 | 0.16697 |
349 | 3454 | FLT4 | 1.06985122 | 0.129807 |
350 | 3394 | HDAC6 | 1.070168765 | 0.050617 |
351 | 3122 | ATSV | 1.071291871 | 0.126675 |
352 | 3169 | NME1 | 1.071353382 | 0.062921 |
353 | 3342 | CCNT1 | 1.07208287 | 0.030624 |
354 | 3523 | NOTCH2 | 1.072235801 | 0.096808 |
355 | 3591 | RALB | 1.072637191 | 0.131285 |
356 | 2970 | AATK | 1.073460682 | 0.079116 |
357 | 3593 | VAV2 | 1.073649235 | 0.087372 |
358 | 3489 | SRC | 1.074621049 | 0.096347 |
359 | 3363 | GART | 1.076380891 | 0.07919 |
360 | 3097 | KIF20A | 1.077741628 | 0.065192 |
361 | 3494 | MAPK4 | 1.077922895 | 0.072549 |
362 | 3114 | PIK3CD | 1.078095752 | 0.118845 |
363 | 2976 | NEK7 | 1.078108286 | 0.543136 |
364 | 3352 | NR3C2 | 1.078524745 | 0.200714 |
365 | 3115 | MDM2 | 1.079408163 | 0.109166 |
366 | 3108 | KIF22 | 1.080326814 | 0.089686 |
367 | 2973 | NEK1 | 1.080546527 | 0.210334 |
368 | 3219 | CENPC1 | 1.080637703 | 0.211586 |
369 | 3583 | JUNB | 1.080828682 | 0.061182 |
370 | 3476 | PRKCD | 1.081421932 | 0.063705 |
371 | 3717 | NTRK2 | 1.08184551 | 0.179359 |
372 | 3760 | CDKL5 | 1.082031957 | 0.122857 |
373 | 3744 | PRKWNK4 | 1.082821695 | 0.041089 |
374 | 3147 | CDKN2A | 1.083174768 | 0.142556 |
375 | 3170 | BLM | 1.083396707 | 0.087103 |
376 | 3390 | NM_080925 | 1.083814073 | 0.187306 |
377 | 3691 | NM_024046 | 1.084007951 | 0.455202 |
378 | 3682 | DYRK1A | 1.085383077 | 0.13164 |
379 | 3338 | CUL4A | 1.085696166 | 0.113752 |
380 | 3445 | BMPR1A | 1.08653048 | 0.217388 |
381 | 3639 | STAT6 | 1.087172241 | 0.240711 |
382 | 3683 | NM_003138 | 1.087765627 | 0.107482 |
383 | 3694 | STK38 | 1.088925769 | 0.15309 |
384 | 3228 | CDC27 | 1.089546461 | 0.230438 |
385 | 2923 | ERN1 | 1.090052682 | 0.160503 |
386 | 3366 | TYMS | 1.090784989 | 0.157841 |
387 | 3816 | NM_017769 | 1.090876067 | 0.170619 |
388 | 3107 | KIF2 | 1.091300875 | 0.082185 |
389 | 3262 | LATS1 | 1.09148938 | 0.058919 |
390 | 3188 | PMS2 | 1.092050213 | 0.140727 |
391 | 3498 | CSNK1A1 | 1.092706943 | 0.059983 |
392 | 3293 | CDC25A | 1.092986402 | 0.099227 |
393 | 3721 | ANKRD3 | 1.093127467 | 0.114101 |
394 | 3793 | MAPRE1 | 1.093414458 | 0.107517 |
395 | 3305 | CDC2L5 | 1.095069991 | 0.058969 |
396 | 3647 | YES1 | 1.095220118 | 0.439175 |
397 | 3324 | CUL5 | 1.095253758 | 0.109464 |
398 | 2965 | NM_014720 | 1.095861428 | 0.295852 |
399 | 3300 | CDC14B | 1.095900812 | 0.053276 |
400 | 3296 | CDKN2C | 1.096728587 | 0.06043 |
401 | 3724 | EPHA7 | 1.096779937 | 0.20814 |
402 | 3165 | FGF2 | 1.099204865 | 0.052442 |
403 | 2928 | IRAK1 | 1.099544846 | 0.11705 |
404 | 3502 | PRKCH | 1.099795802 | 0.076493 |
405 | 3728 | TIE | 1.100408042 | 0.059759 |
406 | 3424 | EZH2 | 1.100414429 | 0.137994 |
407 | 3756 | CDK5RAP2 | 1.10148794 | 0.169172 |
408 | 2920 | EIF2AK3 | 1.101874679 | 0.193517 |
409 | 3556 | RAP1A | 1.102603353 | 0.216629 |
410 | 3214 | CENPF | 1.102666055 | 0.229565 |
411 | 3102 | CKS2 | 1.103490084 | 0.276109 |
412 | 2974 | NEK11 | 1.103575721 | 0.38662 |
413 | 3297 | CCT2 | 1.103974529 | 0.075386 |
414 | 3393 | HDAC2 | 1.104472861 | 0.074707 |
415 | 3568 | PLD1 | 1.104812311 | 0043874 |
416 | 3470 | RPS6KA1 | 1.104927224 | 0.121509 |
417 | 3496 | EIF4EBP2 | 1.105081332 | 0.026061 |
418 | 3432 | PRC1 | 1.105087833 | 0.109514 |
419 | 3446 | PRKCG | 1.105375817 | 0.11356 |
420 | 3512 | TGFBR1 | 1.106970197 | 0.08351 |
421 | 3749 | NM_139021 | 1.107176607 | 0.060956 |
422 | 3807 | SPAG5 | 1.107200152 | 0.190375 |
423 | 3579 | PDZGEF2 | 1.108384492 | 0.106374 |
424 | 3422 | SMC4L1 | 1.108967343 | 0.168462 |
425 | 3830 | NM_013296 | 1.109620231 | 0.124287 |
426 | 3537 | EIF4EBP1 | 1.110833969 | 0.090069 |
427 | 3684 | STK38L | 1.110835442 | 0.127517 |
428 | 3681 | SRPK1 | 1.11126095 | 0.138319 |
429 | 2990 | NM_015112 | 1.111540967 | 0.290052 |
430 | 3605 | FZD4 | 1.111605705 | 0.110001 |
431 | 3477 | FGFR4 | 1.111898761 | 0.065007 |
432 | 3490 | ERBB3 | 1.113605654 | 0.088278 |
433 | 3575 | LATS2 | 1.113869957 | 0.121325 |
434 | 3755 | CDKL3 | 1.114362934 | 0.239022 |
435 | 3205 | NM_139286 | 1.114649942 | 0.243935 |
436 | 3105 | BUB1 | 1.114727935 | 0.21132 |
437 | 3389 | NM_052963 | 1.114830338 | 0.092164 |
438 | 3110 | KIF13A | 1.116195509 | 0.073039 |
439 | 3608 | MAP3K7IP1 | 1.117324513 | 0.193266 |
440 | 2957 | TYK2 | 1.11788043 | 0.120323 |
441 | 2996 | MAPK3 | 1.117972689 | 0.307163 |
442 | 3628 | CTNNBL1 | 1.118548429 | 0.092234 |
443 | 3624 | CTNNB1 | 1.118609166 | 0.170984 |
444 | 3159 | RET | 1.118867767 | 0.029128 |
445 | 3120 | PIK3CB | 1.119135316 | 0.222604 |
446 | 3742 | RHOK | 1.119296716 | 0.166613 |
447 | 3136 | XM_066649 | 1.119463101 | 0.130616 |
448 | 3328 | CCNC | 1.119489673 | 0.067201 |
449 | 3199 | NF2 | 1.119765637 | 0.070805 |
450 | 3309 | CCND2 | 1.121333431 | 0.146937 |
451 | 3143 | NM_017596 | 1.121623368 | 0.07995 |
452 | 3208 | ZW10 | 1.121902285 | 0.144279 |
453 | 3753 | CDK5 | 1.123427629 | 0.130821 |
454 | 3001 | PRKY | 1.125456942 | 0.164937 |
455 | 3729 | RYK | 1.125623162 | 0.196578 |
456 | 3156 | MSH2 | 1.125991819 | 0.128643 |
457 | 3253 | PRKCA | 1.126352597 | 0.097687 |
458 | 3607 | TLE1 | 1.126388877 | 0.255505 |
459 | 3818 | A1338451 | 1.126447243 | 0.085307 |
460 | 3530 | NOTCH1 | 1.127939559 | 0.128155 |
461 | 3141 | NM_145754 | 1.129479267 | 0.026346 |
462 | 3768 | ARAF1 | 1.129705288 | 0.086972 |
463 | 3596 | SHMT1 | 1.129818514 | 0.046177 |
464 | 3653 | NPR2 | 1.129853377 | 0.184709 |
465 | 3640 | STAT5B | 1.132589635 | 0.299667 |
466 | 2924 | STK25 | 1.13270396 | 0.083695 |
467 | 3356 | TUBG1 | 1.133623741 | 0.248371 |
468 | 3008 | SGK2 | 1.135373086 | 0.09569 |
469 | 3499 | GRB2 | 1.135404457 | 0.174583 |
470 | 3506 | XM_095827 | 1.135823602 | 0.058483 |
471 | 3770 | TGFBR2 | 1.136061775 | 0.283098 |
472 | 3441 | PRKCI | 1.137712494 | 0.174946 |
473 | 3609 | FZD3 | 1.138082685 | 0.180803 |
474 | 3370 | AR | 1.139336644 | 0.114355 |
475 | 3126 | KIF3B | 1.139588548 | 0.094914 |
476 | 3508 | KIF25 | 1.140573718 | 0.158738 |
477 | 3233 | ROCK1 | 1.140584559 | 0.236383 |
478 | 2941 | DYRK3 | 1.142052549 | 0.138936 |
479 | 3336 | CDC37 | 1.142173919 | 0.132765 |
480 | 3741 | RPS6KB2 | 1.142253082 | 0.114889 |
481 | 3546 | INPP5D | 1.142646282 | 0.17732 |
482 | 3350 | ADA | 1.14270522 | 0.202027 |
483 | 3759 | NM_006622 | 1.143528436 | 0.053271 |
484 | 3149 | TP53 | 1.144116968 | 0.028664 |
485 | 3310 | CDC34 | 1.145001246 | 0.124753 |
486 | 3267 | CCNH | 1.145203121 | 0.081778 |
487 | 3638 | STAT5A | 1.145284022 | 0.182015 |
488 | 3564 | RALBP1 | 1.145302766 | 0.187726 |
489 | 3360 | RRM2 | 1.145371751 | 0.106232 |
490 | 3662 | LCK | 1.145638747 | 0.091184 |
491 | 3223 | NM_016263 | 1.145667614 | 0.160673 |
492 | 3408 | PIN1 | 1.146539359 | 0.100954 |
493 | 2986 | ACVR2 | 1.146661813 | 0.10512 |
494 | 3304 | CCNE2 | 1.146795654 | 0.102769 |
495 | 2997 | MST1R | 1.147221866 | 0.283163 |
496 | 3194 | RARB | 1.14777913 | 0.330433 |
497 | 3669 | NTRK3 | 1.148222927 | 0.037566 |
498 | 3616 | FZD1 | 1.148473923 | 0.242876 |
499 | 3255 | CDK7 | 1.148553587 | 0.16951 |
500 | 3238 | MAP3K3 | 1.1489897 | 0.067123 |
501 | 3613 | DVL1 | 1.14901778 | 0.082647 |
502 | 3614 | CTNND2 | 1.14937005 | 0.187988 |
503 | 3318 | CUL2 | 1.150267783 | 0.078013 |
504 | 3644 | EPHB1 | 1.15071896 | 0.123257 |
505 | 3567 | SHC1 | 1.151587989 | 0.124227 |
506 | 3116 | KIF5A | 1.152039039 | 0.280422 |
507 | 3148 | LIG1 | 1.152183128 | 0.190895 |
508 | 3765 | CREBBP | 1.154589409 | 0.128712 |
509 | 3232 | KDR | 1.156581097 | 0.11153 |
510 | 3748 | NM_016507 | 1.157187762 | 0.187551 |
511 | 3428 | ECT2 | 1.157383105 | 0.171141 |
512 | 3649 | CAMK2B | 1.157415503 | 0.051472 |
513 | 3426 | TK1 | 1.158048559 | 0.161458 |
514 | 3250 | CHEK2 | 1.158473201 | 0.099737 |
515 | 3636 | STAT2 | 1.158495567 | 0.161875 |
516 | 3187 | WNT7B | 1.158590559 | 0.024217 |
517 | 3505 | STK6 | 1.159146577 | 0.058438 |
518 | 3341 | APLP2 | 1.160801239 | 0.196169 |
519 | 3606 | CREBBP | 1.161326405 | 0.064695 |
520 | 3263 | CDC2 | 1.161570849 | 0.095606 |
521 | 2939 | TLK1 | 1.163052719 | 0.110779 |
522 | 3123 | AKT3 | 1.163145874 | 0.306815 |
523 | 3615 | FZD2 | 1.16322422 | 0.169339 |
524 | 3688 | GUCY2D | 1.164321663 | 0.152801 |
525 | 3379 | NM_032525 | 1.16446975 | 0.074802 |
526 | 3710 | GPRK2L | 1.164900142 | 0.112446 |
527 | 3611 | CTNNAL1 | 1.166257931 | 0.037335 |
528 | 3521 | MET | 1.168013918 | 0.232923 |
529 | 3659 | NM_015978 | 1.169523683 | 0.056392 |
530 | 3582 | GRAP2 | 1.170573492 | 0.055118 |
531 | 3562 | RASD1 | 1.171229699 | 0.101195 |
532 | 3723 | NM_018401 | 1.1718512 | 0.239747 |
533 | 3130 | FRAP1 | 1.172072928 | 0.029779 |
534 | 3772 | RPS6KB1 | 1.172823934 | 0.066518 |
535 | 3333 | CCNT2 | 1.174235642 | 0.214732 |
536 | 3501 | RPS6KA2 | 1.175781534 | 0.193038 |
537 | 3803 | MPHOSPH1 | 1.176011971 | 0.128864 |
538 | 3248 | JAK2 | 1.176020977 | 0.176345 |
539 | 3538 | NFKB2 | 1.176129803 | 0.052353 |
540 | 3732 | CSNK2A2 | 1.177521611 | 0.231267 |
541 | 3730 | TESK1 | 1.177904268 | 0.212526 |
542 | 2989 | ACVR1B | 1.178578161 | 0.217492 |
543 | 3327 | CDC45L | 1.180204158 | 0.299357 |
544 | 3301 | CCNB1 | 1.180864124 | 0.162992 |
545 | 3092 | KIF12 | 1.181937605 | 0.088708 |
546 | 3239 | CDK6 | 1.182157904 | 0.061044 |
547 | 3190 | WNT4 | 1.182697676 | 0.072644 |
548 | 3811 | NM_152524 | 1.183191701 | 0.14187 |
549 | 2940 | DCAMKL1 | 1.184491938 | 0.124698 |
550 | 3761 | WT1 | 1.184547796 | 0.16129 |
551 | 3439 | EGR2 | 1.185671136 | 0.105284 |
552 | 3295 | CDK2AP1 | 1.187045536 | 0.206856 |
553 | 3817 | NM_019013 | 1.187780743 | 0.082116 |
554 | 3754 | CDKL2 | 1.188123077 | 0.122877 |
555 | 3663 | ALS2CR2 | 1.188246404 | 0.140777 |
556 | 3718 | PTK6 | 1.188784586 | 0.067781 |
557 | 3236 | PTK2B | 1.189818532 | 0.352399 |
558 | 3475 | EPHB4 | 1.189888477 | 0.105406 |
559 | 3211 | BUB1B | 1.189896824 | 0.292886 |
560 | 3411 | HIF1A | 1.19069666 | 0.245883 |
561 | 2927 | MAPK13 | 1.190710916 | 0.129633 |
562 | 3264 | CDK3 | 1.191042267 | 0.100335 |
563 | 3207 | MAD1L1 | 1.191232546 | 0.092266 |
564 | 3372 | TUBA8 | 1.191540593 | 0.058385 |
565 | 3349 | IMPDH1 | 1.191967983 | 0.225505 |
566 | 3353 | PGR | 1.192360399 | 0.019529 |
567 | 3252 | NEK2 | 1.192601635 | 0.282445 |
568 | 3515 | PDGFRB | 1.192640873 | 0.057678 |
569 | 3216 | CDC20 | 1.193040181 | 0.077143 |
570 | 2971 | DAPK2 | 1.19306626 | 0.085366 |
571 | 3552 | PDK1 | 1.194218291 | 0.064673 |
572 | 3823 | NM_017779 | 1.194861064 | 0.138396 |
573 | 3528 | TCF3 | 1.195851197 | 0.12389 |
574 | 3201 | RARA | 1.19739521 | 0.087982 |
575 | 2945 | CDC42BPB | 1.19753147 | 0.087566 |
576 | 3634 | BTRC | 1.201076339 | 0.175356 |
577 | 3377 | NM_006088 | 1.202720091 | 0.096411 |
578 | 3781 | SRC | 1.202931814 | 0.331139 |
579 | 3516 | ARHA | 1.203109256 | 0.159342 |
580 | 3700 | AB037782 | 1.204329771 | 0.402706 |
581 | 3699 | NM_032844 | 1.207623266 | 0.248703 |
582 | 2931 | MAP4K3 | 1.211854633 | 0.149673 |
583 | 3189 | MYB | 1.212003569 | 0.117606 |
584 | 3586 | RASA2 | 1.212166142 | 0.210711 |
585 | 3836 | TP53 | 1.212183899 | 0.169152 |
586 | 3206 | ANAPC5 | 1.213545746 | 0.079256 |
587 | 3701 | STK10 | 1.214412753 | 0.23119 |
588 | 3210 | NM_013366 | 1.21450599 | 0.307913 |
589 | 3472 | MAP3K5 | 1.215042561 | 0.128134 |
590 | 3371 | NM_006087 | 1.216059457 | 0.10804 |
591 | 3825 | NM_152562 | 1.216108937 | 0.153345 |
592 | 3106 | KIF9 | 1.217161737 | 0.277445 |
593 | 3249 | MAP2K6 | 1.217382649 | 0.23408 |
594 | 3186 | ETS1 | 1.218428895 | 0.125809 |
595 | 3541 | PKD2 | 1.220374384 | 0.252301 |
596 | 3654 | VRK2 | 1.221266095 | 0.180133 |
597 | 3151 | MLH1 | 1.221977195 | 0.100529 |
598 | 3325 | CDKN1C | 1.222573895 | 0.183555 |
599 | 3774 | CDC45L | 1.223373496 | 0.170335 |
600 | 3354 | RRM1 | 1.224155502 | 0.163218 |
601 | 3225 | NM_013367 | 1.226360075 | 0.377268 |
602 | 3837 | PRKAA1 | 1.226867311 | 0.260099 |
603 | 2930 | ITK | 1.227438086 | 0.134102 |
604 | 3118 | NM_032559 | 1.22825648 | 0.037564 |
605 | 3316 | CCNA1 | 1.228667093 | 0.203935 |
606 | 3651 | VRK1 | 1.229029159 | 0.155208 |
607 | 3368 | top3A | 1.229032423 | 0.14134 |
608 | 3376 | AGA | 1.231363135 | 0.128058 |
609 | 3735 | PRKACB | 1.231534849 | 0.092436 |
610 | 3007 | MAP3K14 | 1.234231675 | 0.17551 |
611 | 3420 | NM_014109 | 1.234890989 | 0.370528 |
612 | 3131 | KIF1B | 1.235185989 | 0.242087 |
613 | 3444 | NEK3 | 1.23520517 | 0.358385 |
614 | 2919 | OSR1 | 1.237086236 | 0.106562 |
615 | 3128 | AKT2 | 1.242407611 | 0.124394 |
616 | 3810 | Al278633 | 1.243126735 | 0.165137 |
617 | 3337 | CDKN1A | 1.244628023 | 0.018797 |
618 | 3091 | KIFC3 | 1.244757733 | 0.153624 |
619 | 3191 | WNT2 | 1.244817311 | 0.187282 |
620 | 3146 | KIF21A | 1.24572579 | 0.041267 |
621 | 3220 | ANAPC11 | 1.246197987 | 0.17988 |
622 | 3785 | GRB2 | 1.248971557 | 0.108491 |
623 | 3195 | CDH1 | 1.250259859 | 0.186152 |
624 | 3500 | SGK | 1.251914788 | 0.059299 |
625 | 3103 | PIK3CA | 1.252248606 | 0.068043 |
626 | 3507 | NM_145754 | 1.252689721 | 0.222951 |
627 | 3565 | RAB2 | 1.253586902 | 0.186552 |
628 | 3462 | TGFBR2 | 1.256459996 | 0.136016 |
629 | 3229 | PRKCL1 | 1.256649876 | 0.153419 |
630 | 3790 | ERBB3 | 1.260353633 | 0.104586 |
631 | 3704 | ACVR2B | 1.261857433 | 0.075994 |
632 | 3340 | CENPH | 1.262786326 | 0.131215 |
633 | 3598 | PCNA | 1.265667779 | 0.116032 |
634 | 2967 | NM_016653 | 1.266923195 | 0.232771 |
635 | 3725 | EPHA4 | 1.267438993 | 0.19056 |
636 | 2932 | MAPKAPK3 | 1.268643945 | 0.025332 |
637 | 3167 | S100A2 | 1.270859999 | 0.069296 |
638 | 2994 | MATK | 1.271154735 | 0.128018 |
639 | 3315 | CCT4 | 1.272355192 | 0.309065 |
640 | 3344 | CDKL1 | 1.272536383 | 0.273155 |
641 | 3689 | BLK | 1.27387895 | 0.218306 |
642 | 3104 | CDK4 | 1.276578446 | 0.161716 |
643 | 3604 | TK2 | 1.277912947 | 0.101801 |
644 | 3209 | MAD2L2 | 1.278038114 | 0.253976 |
645 | 3554 | PIK3R3 | 1.280284314 | 0.228353 |
646 | 3218 | CDC23 | 1.280483947 | 0.334381 |
647 | 3670 | MAP3K10 | 1.280649754 | 0.129166 |
648 | 3532 | NM_019089 | 1.280872331 | 0.138131 |
649 | 3558 | RALA | 1.282343213 | 0.193164 |
650 | 3440 | FGFR3 | 1.283277949 | 0.278946 |
651 | 3779 | CTNNA1 | 1.285066069 | 0.05853 |
652 | 3312 | CUL3 | 1.286086663 | 0.095171 |
653 | 3111 | KIF5B | 1.286155963 | 0.045454 |
654 | 3320 | CCND3 | 1.286427699 | 0.049781 |
655 | 3493 | MAPK9 | 1.286708555 | 0.204254 |
656 | 3463 | TEC | 1.286731353 | 0.116346 |
657 | 3198 | ICAM1 | 1.287087211 | 0.105164 |
658 | 2933 | MAP4K5 | 1.288848714 | 0.19588 |
659 | 2995 | PTK2 | 1.289781227 | 0.122006 |
660 | 3637 | STAT4 | 1.291478071 | 0.126765 |
661 | 3089 | KIFC1 | 1.292296631 | 0.104185 |
662 | 3330 | CDK9 | 1.293189989 | 0.200332 |
663 | 3588 | RHEB | 1.295786915 | 0.113922 |
664 | 3589 | SOS1 | 1.300834959 | 0.028846 |
665 | 3418 | CENPA | 1.300851356 | 0.224648 |
666 | 3314 | CCNG1 | 1.302132075 | 0.167018 |
667 | 3697 | CAMK2G | 1.305521288 | 0.141582 |
668 | 3620 | AXIN2 | 1.306881235 | 0.175725 |
669 | 2921 | RPS6KA5 | 1.307895788 | 0.116976 |
670 | 3157 | NF1 | 1.312364005 | 0.21979 |
671 | 3172 | PLAU | 1.314219765 | 0.221395 |
672 | 3221 | top3B | 1.316767724 | 0.153732 |
673 | 3529 | DTX1 | 1.317104131 | 0.100042 |
674 | 3520 | NRAS | 1.318162379 | 0.337798 |
675 | 3138 | KIF17 | 1.319021332 | 0.047239 |
676 | 3466 | JAK3 | 1.324590857 | 0.341923 |
677 | 3447 | PRKCM | 1.325852782 | 0.090164 |
678 | 3396 | HDAC10 | 1.327036067 | 0.095421 |
679 | 3405 | HDAC8 | 1.328986383 | 0.137456 |
680 | 2956 | PRKCL2 | 1.329759987 | 0.277544 |
681 | 3771 | PIK3CA | 1.330927854 | 0.318605 |
682 | 3100 | GSK3B | 1.332661843 | 0.172085 |
683 | 3140 | XM_089006 | 1.334634688 | 0.210199 |
684 | 3417 | HDAC3 | 1.334739136 | 0.213735 |
685 | 2912 | MPHOSPH1 | 1.335606817 | 0.192246 |
686 | 3453 | MAP2K2 | 1.337178184 | 0.231133 |
687 | 3777 | ABL1 | 1.337946355 | 0.13381 |
688 | 2991 | NPR1 | 1.339668528 | 0.213719 |
689 | 3234 | CDK2 | 1.341378632 | 0.327603 |
690 | 3617 | CTNNBIP1 | 1.344129969 | 0.172119 |
691 | 3217 | NM_014885 | 1.346642104 | 0.37578 |
692 | 3632 | WISP1 | 1.34798621 | 0.267584 |
693 | 3404 | PPARG | 1.350608226 | 0.328799 |
694 | 3834 | CHEK1 | 1.353682807 | 0.177332 |
695 | 3244 | PRKCZ | 1.354506447 | 0.423569 |
696 | 3242 | PRKCB1 | 1.356110177 | 0.177856 |
697 | 2998 | MAPK7 | 1.357915027 | 0.320918 |
698 | 3227 | NUMA1 | 1.358033567 | 0.336206 |
699 | 3676 | MAP4K1 | 1.360624202 | 0.35665 |
700 | 3087 | PTEN | 1.361043077 | 0.221803 |
701 | 3734 | BMPR1B | 1.362751745 | 0.19663 |
702 | 3569 | RASGRP2 | 1.362852713 | 0.083746 |
703 | 2953 | MAPK11 | 1.367417583 | 0.335411 |
704 | 3355 | GUK1 | 1.368854888 | 0.121328 |
705 | 3713 | PRKG2 | 1.370762753 | 0.096281 |
706 | 3415 | HSPCA | 1.373102391 | 0.311637 |
707 | 3212 | NM_022662 | 1.373845206 | 0.3643 |
708 | 3789 | ELK1 | 1.378293297 | 0.119276 |
709 | 3395 | HDAC5 | 1.380918509 | 0.316118 |
710 | 3448 | NM_016231 | 1.382981639 | 0.250346 |
711 | 3737 | NM_016457 | 1.384393078 | 0.35016 |
712 | 3456 | FLT1 | 1.387001071 | 0.117573 |
713 | 3696 | NM_016281 | 1.392513247 | 0.179922 |
714 | 3124 | KIF4A | 1.392774473 | 0.395931 |
715 | 3451 | MAP3K4 | 1.39359615 | 0.215328 |
716 | 3738 | PRKWNK3 | 1.395197042 | 0.116947 |
717 | 3719 | BMPR2 | 1.395676978 | 0.083941 |
718 | 3429 | MCM6 | 1.399624047 | 0.422475 |
719 | 3243 | NM_004203 | 1.401278663 | 0.303675 |
720 | 3660 | DMPK | 1.402604745 | 0.203011 |
721 | 3084 | KIF14 | 1.405667444 | 0.022909 |
722 | 3574 | SH3KBP1 | 1.408096057 | 0.080055 |
723 | 3137 | KIF26A | 1.410397298 | 0.209271 |
724 | 3671 | STK4 | 1.410482157 | 0.306699 |
725 | 3202 | MCC | 1.410775773 | 0.285878 |
726 | 3134 | XM_170783 | 1.415482722 | 0.226917 |
727 | 3204 | CDC16 | 1.415780291 | 0.221962 |
728 | 3121 | PIK3C2A | 1.415950012 | 0.152356 |
729 | 3321 | CDKN3 | 1.416176725 | 0.03826 |
730 | 2951 | AJ311798 | 1.416769938 | 0.177239 |
731 | 3504 | PIK3CB | 1.417592955 | 0.174632 |
732 | 2955 | PAK1 | 1.418341722 | 0.07362 |
733 | 3612 | TCF1 | 1.421215206 | 0.099637 |
734 | 3655 | CAMK2D | 1.422993988 | 0.227042 |
735 | 3135 | XM_064050 | 1.42777471 | 0.205042 |
736 | 3400 | BCL2 | 1.432342001 | 0.20388 |
737 | 3794 | WASL | 1.432665996 | 0.093756 |
738 | 3667 | NM_016542 | 1.434249905 | 0.174478 |
739 | 3407 | HDAC9 | 1.437540011 | 0.194891 |
740 | 3430 | STMN1 | 1.437943227 | 0.313077 |
741 | 3698 | ADRBK2 | 1.440932223 | 0.248217 |
742 | 3547 | FOXO1A | 1.461102151 | 0.113568 |
743 | 3265 | RAF1 | 1.463315846 | 0.191102 |
744 | 3690 | PRKAA2 | 1.470696795 | 0.233827 |
745 | 3510 | CDK4 | 1.487289818 | 0.089318 |
746 | 3254 | CSF1R | 1.487946096 | 0.480379 |
747 | 3622 | FZD9 | 1.49526423 | 0.086599 |
748 | 3544 | IRS1 | 1.495662211 | 0.040512 |
749 | 2948 | MYO3A | 1.4970851 | 0.081259 |
750 | 3467 | MAP2K7 | 1.497928287 | 0.281253 |
751 | 3096 | AKT1 | 1.498269053 | 0.114084 |
752 | 2968 | STK17B | 1.504346366 | 0.413498 |
753 | 3402 | HDAC4 | 1.508119762 | 0.213089 |
754 | 3764 | NOTCH4 | 1.510551197 | 0.081586 |
755 | 3621 | CTNNA2 | 1.511056295 | 0.111649 |
756 | 3168 | DCC | 1.513409582 | 0.192599 |
757 | 2701 | 2701 | 1.51348211 | 0.083391 |
758 | 3629 | AXIN1 | 1.520734118 | 0.174178 |
759 | 3361 | IMPDH2 | 1.523631011 | 0.067873 |
760 | 3129 | STK6 | 1.527103437 | 0.134504 |
761 | 3679 | CLK2 | 1.53626119 | 0.44738 |
762 | 3709 | X95425 | 1.537827387 | 0.437676 |
763 | 2962 | MAP4K2 | 1.547893113 | 0.329021 |
764 | 3442 | ERBB4 | 1.551008953 | 0.146803 |
765 | 3247 | NM_018492 | 1.552802846 | 0.137033 |
766 | 3720 | AB002301 | 1.553258633 | 0.313891 |
767 | 3584 | RASAL2 | 1.563405608 | 0.137336 |
768 | 3299 | CUL1 | 1.589913659 | 0.169909 |
769 | 3522 | KRAS2 | 1.590661017 | 0.06841 |
770 | 3590 | ARHGEF2 | 1.597950927 | 0.252526 |
771 | 3406 | TERT | 1.600169172 | 0.094096 |
772 | 3259 | MAPK8 | 1.601990057 | 0.333883 |
773 | 3369 | NM_007027 | 1.605090071 | 0.162172 |
774 | 3787 | FZD4 | 1.621497881 | 0.053639 |
775 | 2929 | CHUK | 1.646057679 | 0.111716 |
776 | 3468 | ABL2 | 1.652007602 | 0.180802 |
777 | 2988 | FRK | 1.653152871 | 0.298882 |
778 | 3758 | RAD51L1 | 1.662423293 | 0.135675 |
779 | 3531 | NM_021170 | 1.666989154 | 0.094206 |
780 | 3155 | ATR | 1.680571715 | 0.388687 |
781 | 3747 | GSK3A | 1.688637713 | 0.38953 |
782 | 3144 | KIF4B | 1.695873891 | 0.467258 |
783 | 3235 | CHEK1 | 1.697910825 | 0.356224 |
784 | 3313 | CCNG2 | 1.703651114 | 0.216266 |
785 | 3004 | MAP3K1 | 1.721492222 | 0.376438 |
786 | 3619 | FRAT1 | 1.761446915 | 0.292031 |
787 | 3192 | WNT1 | 1.765037748 | 0.394063 |
788 | 3673 | DDR1 | 1.770053978 | 0.2338 |
789 | 3358 | top2B | 1.800293702 | 0.195754 |
790 | 2981 | ALK | 1.84348901 | 0.208338 |
791 | 2958 | PRKACA | 1.889142934 | 0.494773 |
792 | 3152 | APC | 1.894694006 | 0.191358 |
793 | 3712 | RPS6KA6 | 1.957145081 | 0.421292 |
794 | 3436 | BRAF | 1.999825737 | 1.173208 |
795 | 3727 | GPRK6 | 2.044605743 | 0.256806 |
796 | 3780 | MCM3 | 2.062893191 | 0.187038 |
797 | 3329 | CDC42 | 2.131693629 | 0.483392 |
798 | 3095 | KIF2C | 2.163834467 | 0.289685 |
799 | 3098 | CENPE | 2.170456559 | 0.120025 |
800 | 3331 | CDC25B | 2.199340751 | 0.484716 |
801 | 3706 | C20orf97 | 2.377822809 | 0.678329 |
802 | 3580 | ELK1 | 2.456195789 | 0.434043 |
803 | 3241 | WEE1 | 2.66755235 | 0.625231 |
804 | 3642 | EPHB3 | 2.758093154 | 0.565256 |
805 | 3158 | BRCA1 | 2.878071685 | 0.418358 |
806 | 3150 | BRCA2 | 11.61633698 | 1.101248 |
The average sensitization multiple that Table II B camptothecine causes
Gene I | BIOID | Gene | The sensitization multiple |
1 | 63 | M15077 | 1 |
2 | 2514 | PLK | 0.029197 |
3 | 2540 | 2540 | #DIV/01 |
4 | 2541 | 2541 | 0.860453 |
5 | 2701 | 2701 | 0.034091 |
6 | 2702 | 2702 | 0.432441 |
7 | 3391 | PLK | 0.052632 |
8 | 3534 | PLK | 0.083815 |
9 | 3099 | PLK | 0.090142 |
10 | 3006 | PLK | 0.09146 |
11 | 3266 | PLK | 0.096774 |
12 | 3433 | PLK | 0.13 |
13 | 3322 | CCNA2 | 0.264029 |
14 | 3154 | MADH4 | 0.361653 |
15 | 3518 | NFKB1 | 0.372726 |
16 | 3600 | RRM2B | 0.381056 |
17 | 3184 | TSG101 | 0.432287 |
18 | 3348 | DCK | 0.446467 |
19 | 3332 | CDC5L | 0.451264 |
20 | 3812 | CDCA8 | 0.453177 |
21 | 3423 | NM_006101 | 0.478261 |
22 | 3464 | INSR | 0.480578 |
23 | 2961 | PIM1 | 0.51581 |
24 | 3661 | FGR | 0.517647 |
25 | 3171 | VHL | 0.524194 |
26 | 3809 | CDCA3 | 0.529046 |
27 | 3525 | NOTCH4 | 0.534058 |
28 | 3093 | PIK3CG | 0.557692 |
29 | 3740 | STK35 | 0.55782 |
30 | 3435 | FLT3 | 0.56 |
31 | 3805 | C20orf1 | 0.564035 |
32 | 3219 | CENPC1 | 0.575465 |
33 | 3003 | FER | 0.579832 |
34 | 3183 | NM_005200 | 0.580153 |
35 | 3374 | POLR2A | 0.583796 |
36 | 3601 | POLE | 0.588331 |
37 | 3112 | KNSL7 | 0.597685 |
38 | 3489 | SRC | 0.606833 |
39 | 3478 | EPHA2 | 0.608258 |
40 | 3422 | SMC4L1 | 0.608696 |
41 | 3357 | PRIM2A | 0.611218 |
42 | 3262 | LATS1 | 0.613321 |
43 | 2987 | RNASEL | 0.617089 |
44 | 3123 | AKT3 | 0.618574 |
45 | 3687 | CAMK4 | 0.61913 |
46 | 3303 | CDKN2D | 0.625741 |
47 | 2966 | NM_033266 | 0.627321 |
48 | 3226 | RBX1 | 0.632166 |
49 | 3509 | KIF21A | 0.634426 |
50 | 2999 | FES | 0.634596 |
51 | 3517 | MYC | 0.637624 |
52 | 3592 | SOS2 | 0.640343 |
53 | 3139 | XM_095827 | 0.642535 |
54 | 3105 | BUB1 | 0.643861 |
55 | 3397 | BUB3 | 0.644077 |
56 | 3267 | CCNH | 0.64503 |
57 | 2975 | NEK4 | 0.645485 |
58 | 3766 | S100A2 | 0.646739 |
59 | 2936 | SGKL | 0.64695 |
60 | 3524 | NOTCH3 | 0.647109 |
61 | 3806 | C10orf3 | 0.64878 |
62 | 3448 | NM_016231 | 0.654135 |
63 | 3461 | KIT | 0.662033 |
64 | 3501 | RPS6KA2 | 0.667039 |
65 | 3494 | MAPK4 | 0.668898 |
66 | 3251 | ABL1 | 0.675159 |
67 | 3103 | PIK3CA | 0.679543 |
68 | 3572 | RASA3 | 0.681105 |
69 | 3246 | RPS6KB1 | 0.681548 |
70 | 3230 | MAP2K1 | 0.683985 |
71 | 3733 | MYLK2 | 0.684534 |
72 | 3491 | PRKCE | 0.685882 |
73 | 2982 | CDC2L2 | 0.687831 |
74 | 3542 | PPP2CA | 0.690237 |
75 | 3350 | ADA | 0.692046 |
76 | 3651 | VRK1 | 0.692308 |
77 | 2937 | NM_025052 | 0.693089 |
78 | 3007 | MAP3K14 | 0.694169 |
79 | 3751 | BCR | 0.694278 |
80 | 3410 | RPS27 | 0.695238 |
81 | 3240 | MAPK12 | 0.696498 |
82 | 2949 | MYO3B | 0.698039 |
83 | 3413 | KIF23 | 0.701413 |
84 | 3773 | WNT2 | 0.702997 |
85 | 3762 | MCC | 0.706533 |
86 | 3731 | CSNK1E | 0.707275 |
87 | 3778 | VHL | 0.707386 |
88 | 3476 | PRKCD | 0.708251 |
89 | 3754 | CDKL2 | 0.712665 |
90 | 3741 | RPS6KB2 | 0.715261 |
91 | 3744 | PRKWNK4 | 0.716089 |
92 | 3148 | LIG1 | 0.71816 |
93 | 2964 | RIPK2 | 0.71873 |
94 | 3486 | RPS6KA3 | 0.71875 |
95 | 3772 | RPS6KB1 | 0.722315 |
96 | 3193 | FGF3 | 0.723179 |
97 | 3363 | GART | 0.723732 |
98 | 3438 | DTR | 0.725061 |
99 | 3351 | ESR1 | 0.725395 |
100 | 3416 | IKBKE | 0.726044 |
101 | 2972 | KSR | 0.727171 |
102 | 3326 | CCT7 | 0.727769 |
103 | 3648 | CLK1 | 0.728232 |
104 | 3401 | HDAC1 | 0.728268 |
105 | 3498 | CSNK1A1 | 0.728714 |
106 | 2976 | NEK7 | 0.729805 |
107 | 3347 | top1 | 0.731826 |
108 | 3236 | PTK2B | 0.736339 |
109 | 3256 | CDC2L1 | 0.738272 |
110 | 3606 | CREBBP | 0.738487 |
111 | 3657 | PCTK1 | 0.739866 |
112 | 3452 | JAK1 | 0.745847 |
113 | 3250 | CHEK2 | 0.745919 |
114 | 3200 | REL | 0.746919 |
115 | 3403 | AURKC | 0.747841 |
116 | 3663 | ALS2CR2 | 0.749671 |
117 | 3208 | ZW10 | 0.75 |
118 | 3647 | YES1 | 0.750637 |
119 | 3466 | JAK3 | 0.750708 |
120 | 3196 | ARH1 | 0.75402 |
121 | 3757 | CLK4 | 0.757793 |
122 | 3434 | DDX6 | 0.758671 |
123 | 3460 | CSK | 0.759454 |
124 | 3722 | TLK2 | 0.761568 |
125 | 3306 | CDC14A | 0.761859 |
126 | 3412 | KIF25 | 0.761959 |
127 | 2926 | AF172264 | 0.762856 |
128 | 3382 | TUBB | 0.763294 |
129 | 2965 | NM_014720 | 0.763727 |
130 | 3625 | CTBP2 | 0.763827 |
131 | 3702 | MAP3K13 | 0.764125 |
132 | 3650 | NM_025195 | 0.764957 |
133 | 3323 | CDK5R1 | 0.765293 |
134 | 3653 | NPR2 | 0.765609 |
135 | 2997 | MST1R | 0.767068 |
136 | 3658 | STK18 | 0.768411 |
137 | 3739 | NM_017886 | 0.768662 |
138 | 2993 | SRMS | 0.768678 |
139 | 3166 | PMS1 | 0.769717 |
140 | 3775 | NOTCH1 | 0.770983 |
141 | 3469 | AAK1 | 0.772082 |
142 | 3833 | ATR | 0.772423 |
143 | 3211 | BUB1B | 0.773389 |
144 | 3557 | JUND | 0.773496 |
145 | 3179 | PDGFB | 0.777522 |
146 | 3674 | CSNK1D | 0.779923 |
147 | 3566 | JUN | 0.780371 |
148 | 3341 | APLP2 | 0.781888 |
149 | 3188 | PMS2 | 0.785359 |
150 | 3633 | CTBP1 | 0.786631 |
151 | 2923 | ERN1 | 0.787194 |
152 | 3086 | KIF11 | 0.787201 |
153 | 3688 | GUCY2D | 0.787284 |
154 | 3605 | FZD4 | 0.787879 |
155 | 3640 | STAT5B | 0.789018 |
156 | 2974 | NEK11 | 0.791024 |
157 | 3473 | DAPK1 | 0.791285 |
158 | 3376 | AGA | 0.791586 |
159 | 3263 | CDC2 | 0.792593 |
160 | 3475 | EPHB4 | 0.797463 |
161 | 3346 | CCNK | 0.79871 |
162 | 3298 | CCNE1 | 0.800418 |
163 | 3359 | TUBA1 | 0.801205 |
164 | 3609 | FZD3 | 0.806613 |
165 | 3201 | RARA | 0.808157 |
166 | 3394 | HDAC6 | 0.810106 |
167 | 3770 | TGFBR2 | 0.810897 |
168 | 3258 | MOS | 0.811566 |
169 | 3541 | PKD2 | 0.811594 |
170 | 3822 | GTSE1 | 0.814495 |
171 | 3450 | MAP3K2 | 0.81592 |
172 | 3577 | RAB2L | 0.816 |
173 | 3203 | ITGA5 | 0.817391 |
174 | 3838 | PRKAA2 | 0.821543 |
175 | 3085 | KIF5C | 0.82316 |
176 | 3477 | FGFR4 | 0.824427 |
177 | 3573 | NM_016848 | 0.824468 |
178 | 3836 | TP53 | 0.825022 |
179 | 3782 | SOS1 | 0.825161 |
180 | 3366 | TYMS | 0.828914 |
181 | 3381 | POLR2B | 0.828921 |
182 | 3710 | GPRK2L | 0.830756 |
183 | 2934 | IRAK2 | 0.830809 |
184 | 3364 | HPRT1 | 0.831103 |
185 | 3182 | MYCN | 0.831349 |
186 | 3783 | KRAS2 | 0.831863 |
187 | 3113 | CNK | 0.834672 |
188 | 3835 | CHEK2 | 0.836402 |
189 | 3680 | CLK3 | 0.836728 |
190 | 3131 | KIF1B | 0.83697 |
191 | 3088 | KIF13B | 0.838299 |
192 | 3581 | RASGRP1 | 0.839735 |
193 | 3829 | KIF2C | 0.840215 |
194 | 3380 | TUBG2 | 0.840866 |
195 | 3334 | CUL4B | 0.842773 |
196 | 3746 | HUNK | 0.84279 |
197 | 2921 | RPS6KA5 | 0.845122 |
198 | 3769 | PLAU | 0.845466 |
199 | 2984 | EPHB6 | 0.847067 |
200 | 3814 | HMMR | 0.850166 |
201 | 3623 | CTNND1 | 0.850309 |
202 | 3444 | NEK3 | 0.851852 |
203 | 2935 | MAPK6 | 0.852713 |
204 | 2996 | MAPK3 | 0.853188 |
205 | 2969 | NM_014916 | 0.856081 |
206 | 3120 | PIK3CB | 0.856195 |
207 | 3107 | KIF2 | 0.856252 |
208 | 3502 | PRKCH | 0.856893 |
209 | 3763 | NM_016231 | 0.858333 |
210 | 3419 | RFC4 | 0.858657 |
211 | 3639 | STAT6 | 0.858685 |
212 | 2930 | ITK | 0.860156 |
213 | 3124 | KIF4A | 0.860439 |
214 | 3209 | MAD2L2 | 0.860811 |
215 | 3832 | ATM | 0.861555 |
216 | 3774 | CDC45L | 0.862319 |
217 | 3342 | CCNT1 | 0.86272 |
218 | 3430 | STMN1 | 0.864508 |
219 | 3802 | NOTCH3 | 0.865116 |
220 | 3309 | CCND2 | 0.865741 |
221 | 3411 | HIF1A | 0.867769 |
222 | 3717 | NTRK2 | 0.867864 |
223 | 3465 | EPHA1 | 0.867876 |
224 | 3795 | NR4A2 | 0.867991 |
225 | 3659 | NM_015978 | 0.868205 |
226 | 3643 | DDR2 | 0.868618 |
227 | 3392 | BIRC5 | 0.869293 |
228 | 3786 | FRAP1 | 0.870607 |
229 | 3297 | CCT2 | 0.872024 |
230 | 2991 | NPR1 | 0.872727 |
231 | 3318 | CUL2 | 0.87438 |
232 | 3293 | CDC25A | 0.875 |
233 | 3421 | ORC6L | 0.875341 |
234 | 3454 | FLT4 | 0.875663 |
235 | 2950 | NEK6 | 0.876961 |
236 | 3815 | MAPRE2 | 0.877732 |
237 | 3831 | CLSPN | 0.878064 |
238 | 3232 | KDR | 0.878378 |
239 | 3709 | X95425 | 0.879358 |
240 | 2929 | CHUK | 0.881491 |
241 | 3378 | NM_006009 | 0.882129 |
242 | 2952 | PRKG1 | 0.883408 |
243 | 3776 | NOTCH2 | 0.88366 |
244 | 3356 | TUBG1 | 0.884709 |
245 | 3308 | CDKN2B | 0.885077 |
246 | 2967 | NM_016653 | 0.886094 |
247 | 3591 | RALB | 0.889362 |
248 | 3635 | STAT1 | 0.889881 |
249 | 3530 | NOTCH1 | 0.890111 |
250 | 3750 | CAMK2A | 0.891525 |
251 | 3523 | NOTCH2 | 0.893064 |
252 | 2980 | RIPK1 | 0.894417 |
253 | 3249 | MAP2K6 | 0.895216 |
254 | 3589 | SOS1 | 0.895558 |
255 | 3587 | VAV3 | 0.896552 |
256 | 2968 | STK17B | 0.899438 |
257 | 3505 | STK6 | 0.899549 |
258 | 3526 | HES6 | 0.899892 |
259 | 3261 | ERBB2 | 0.904662 |
260 | 3252 | NEK2 | 0.904873 |
261 | 3426 | TK1 | 0.906569 |
262 | 3328 | CCNC | 0.909091 |
263 | 3470 | RPS6KA1 | 0.909627 |
264 | 3798 | ACTR2 | 0.910468 |
265 | 3595 | FEN1 | 0.910569 |
266 | 3597 | SHMT2 | 0.911368 |
267 | 3362 | NR3C1 | 0.911404 |
268 | 3257 | IGF1R | 0.911442 |
269 | 3665 | PAK4 | 0.913313 |
270 | 3678 | PFTK1 | 0.913673 |
271 | 3344 | CDKL1 | 0.913907 |
272 | 3302 | CDK8 | 0.913988 |
273 | 3536 | PIK3C3 | 0.916089 |
274 | 3685 | LTK | 0.917246 |
275 | 3749 | NM_139021 | 0.917681 |
276 | 3268 | CDC25C | 0.919654 |
277 | 3743 | RAGE | 0.922602 |
278 | 3414 | HDAC7A | 0.925495 |
279 | 3162 | MADH2 | 0.927277 |
280 | 3429 | MCM6 | 0.928214 |
281 | 3682 | DYRK1A | 0.928261 |
282 | 3585 | GAB1 | 0.928513 |
283 | 3549 | PIK3R2 | 0.930054 |
284 | 3233 | ROCK1 | 0.930818 |
285 | 3315 | CCT4 | 0.931751 |
286 | 2990 | NM_015112 | 0.933149 |
287 | 3409 | TTK | 0.934641 |
288 | 3237 | CDC7 | 0.938429 |
289 | 2960 | LYN | 0.938849 |
290 | 3664 | STK17A | 0.93923 |
291 | 2931 | MAP4K3 | 0.939649 |
292 | 3693 | NEK9 | 0.939894 |
293 | 3694 | STK38 | 0.941537 |
294 | 3000 | BMX | 0.94164 |
295 | 3445 | BMPR1A | 0.944154 |
296 | 3207 | MAD1L1 | 0.945191 |
297 | 3714 | NM_013355 | 0.947122 |
298 | 3652 | PDGFRA | 0.947533 |
299 | 3631 | WISP2 | 0.948783 |
300 | 3799 | ACTR3 | 0.949315 |
301 | 2912 | MPHOSPH1 | 0.95053 |
302 | 3142 | KIF25 | 0.950655 |
303 | 3755 | CDKL3 | 0.950839 |
304 | 3231 | ILK | 0.951 |
305 | 3155 | ATR | 0.951613 |
306 | 3646 | NM_005781 | 0.952566 |
307 | 2979 | PAK2 | 0.953323 |
308 | 3296 | CDKN2C | 0.954784 |
309 | 2983 | GUCY2C | 0.956701 |
310 | 3497 | EPHA8 | 0.958146 |
311 | 3163 | THRA | 0.959354 |
312 | 3471 | MAPK10 | 0.960665 |
313 | 2940 | DCAMKL1 | 0.963487 |
314 | 3593 | VAV2 | 0.963865 |
315 | 3398 | HDAC11 | 0.964784 |
316 | 3752 | CCNB3 | 0.964824 |
317 | 3641 | TYRO3 | 0.965291 |
318 | 3195 | CDH1 | 0.96542 |
319 | 3552 | PDK1 | 0.96695 |
320 | 3132 | KIF23 | 0.970496 |
321 | 2951 | AJ311798 | 0.971591 |
322 | 3214 | CENPF | 0.974453 |
323 | 3436 | BRAF | 0.97619 |
324 | 3104 | CDK4 | 0.976337 |
325 | 2959 | PIM2 | 0.977075 |
326 | 3228 | CDC27 | 0.978723 |
327 | 3570 | RALGDS | 0.979927 |
328 | 3826 | NM_015694 | 0.981405 |
329 | 3788 | PRKCE | 0.983254 |
330 | 2954 | ROCK2 | 0.983541 |
331 | 3539 | PLCG2 | 0.985447 |
332 | 3732 | CSNK2A2 | 0.985667 |
333 | 3759 | NM_006622 | 0.98881 |
334 | 2998 | MAPK7 | 0.993015 |
335 | 3352 | NR3C2 | 0.996058 |
336 | 3488 | AURKB | 0.996508 |
337 | 3130 | FRAP1 | 0.996898 |
338 | 3691 | NM_024046 | 0.997251 |
339 | 3683 | NM_003138 | 0.998179 |
340 | 3569 | RASGRP2 | 1.000543 |
341 | 2920 | EIF2AK3 | 1.005703 |
342 | 3365 | PRIM1 | 1.006112 |
343 | 3462 | T6FBR2 | 1.00726 |
344 | 3513 | ROS1 | 1.009016 |
345 | 3102 | CKS2 | 1.013052 |
346 | 2945 | CDC42BPB | 1.01398 |
347 | 3656 | PRKCN | 1.016229 |
348 | 3726 | MAPKAPK2 | 1.016458 |
349 | 3002 | CRKL | 1.01909 |
350 | 3670 | MAP3K10 | 1.01919 |
351 | 3767 | FZD3 | 1.019811 |
352 | 3645 | CASK | 1.020319 |
353 | 3707 | TXK | 1.022666 |
354 | 3455 | MAP2K4 | 1.023218 |
355 | 3372 | TUBA8 | 1.025814 |
356 | 3540 | PPP2CB | 1.027826 |
357 | 3690 | PRKAA2 | 1.029902 |
358 | 3307 | CDC6 | 1.03012 |
359 | 3495 | FGFR2 | 1.032093 |
360 | 3485 | DHX8 | 1.032492 |
361 | 3696 | NM_016281 | 1.038149 |
362 | 3716 | ULK1 | 1.039167 |
363 | 3265 | RAF1 | 1.039442 |
364 | 3161 | WT1 | 1.039655 |
365 | 3215 | MAD2L1 | 1.039783 |
366 | 3415 | HSPCA | 1.040186 |
367 | 3127 | MAPK1 | 1.040277 |
368 | 3686 | MAP3K8 | 1.040303 |
369 | 3490 | ERBB3 | 1.040323 |
370 | 3441 | PRKCI | 1.042373 |
371 | 3115 | MDM2 | 1.04276 |
372 | 3264 | CDK3 | 1.044285 |
373 | 3147 | CDKN2A | 1.045872 |
374 | 3568 | PLD1 | 1.048696 |
375 | 3559 | RAP1GDS1 | 1.05 |
376 | 2928 | IRAK1 | 1.050577 |
377 | 3197 | ARHB | 1.052064 |
378 | 3785 | GRB2 | 1.052525 |
379 | 3248 | JAK2 | 1.053539 |
380 | 3199 | NF2 | 1.053654 |
381 | 2992 | PRKR | 1.055468 |
382 | 3516 | ARHA | 1.058051 |
383 | 3449 | TBK1 | 1.059537 |
384 | 2953 | MAPK11 | 1.059656 |
385 | 3164 | MYCL1 | 1.060646 |
386 | 3745 | CAMKK2 | 1.061685 |
387 | 3324 | CUL5 | 1.062571 |
388 | 3243 | NM_004203 | 1.062998 |
389 | 3187 | WNT7B | 1.063935 |
390 | 3459 | EGFR | 1.066553 |
391 | 3239 | CDK6 | 1.067257 |
392 | 3170 | BLM | 1.068402 |
393 | 2943 | DYRK2 | 1.06862 |
394 | 3320 | CCND3 | 1.070018 |
395 | 3369 | NM_007027 | 1.071887 |
396 | 3624 | CTNNB1 | 1.072588 |
397 | 3500 | SGK | 1.074011 |
398 | 3101 | MAPK14 | 1.074871 |
399 | 3408 | PIN1 | 1.075614 |
400 | 2924 | STK25 | 1.076046 |
401 | 3548 | RAC1 | 1.07851 |
402 | 3676 | MAP4K1 | 1.079121 |
403 | 3698 | ADRBK2 | 1.079569 |
404 | 3301 | CCNB1 | 1.080243 |
405 | 2925 | FYN | 1.081081 |
406 | 3565 | RAB2 | 1.081968 |
407 | 2977 | R1PK3 | 1.082037 |
408 | 3810 | AI278633 | 1.084388 |
409 | 3796 | ARHGEF6 | 1.084848 |
410 | 3116 | KIF5A | 1.086755 |
411 | 3590 | ARHGEF2 | 1.088083 |
412 | 3679 | CLK2 | 1.088737 |
413 | 3119 | CDKN1B | 1.089067 |
414 | 3367 | DHFR | 1.092319 |
415 | 3797 | ARHGEF9 | 1.092391 |
416 | 3405 | HDAC8 | 1.096856 |
417 | 2957 | TYK2 | 1.099156 |
418 | 3091 | KIFC3 | 1.10008 |
419 | 3546 | INPP5D | 1.102828 |
420 | 3227 | NUMA1 | 1.104478 |
421 | 3181 | ST5 | 1.104782 |
422 | 3807 | SPAG5 | 1.105317 |
423 | 3090 | KIF3C | 1.10597 |
424 | 3343 | CENPJ | 1.107383 |
425 | 3245 | AXL | 1.108766 |
426 | 3097 | KIF20A | 1.108842 |
427 | 3360 | RRM2 | 1.109827 |
428 | 3349 | IMPDH1 | 1.111043 |
429 | 3474 | CSNK2A1 | 1.111842 |
430 | 3616 | FZD1 | 1.11295 |
431 | 3620 | AXIN2 | 1.113386 |
432 | 2995 | PTK2 | 1.115385 |
433 | 3634 | BTRC | 1.117674 |
434 | 3504 | PIK3CB | 1.118194 |
435 | 3561 | FOS | 1.118649 |
436 | 3618 | DVL2 | 1.12 |
437 | 3537 | EIF4EBP1 | 1.121316 |
438 | 3550 | PLCG1 | 1.121971 |
439 | 3443 | EPS8 | 1.122744 |
440 | 3370 | AR | 1.123767 |
441 | 3543 | PDK2 | 1.12548 |
442 | 3122 | ATSV | 1.127371 |
443 | 3167 | S100A2 | 1.127907 |
444 | 3596 | SHMT1 | 1.128114 |
445 | 3811 | NM_152524 | 1.129555 |
446 | 3779 | CTNNA1 | 1.129565 |
447 | 3312 | CUL3 | 1.133047 |
448 | 2963 | MAP3K11 | 1.133758 |
449 | 2942 | TTN | 1.133889 |
450 | 3790 | ERBB3 | 1.135274 |
451 | 3094 | KIF3A | 1.13729 |
452 | 3545 | IRS2 | 1.139283 |
453 | 3305 | CDC2L5 | 1.140753 |
454 | 3748 | NM_016507 | 1.140961 |
455 | 3614 | CTNND2 | 1.141748 |
456 | 3437 | FGFR1 | 1.143284 |
457 | 3389 | NM_052963 | 1.145845 |
458 | 3213 | NM_016238 | 1.145939 |
459 | 3533 | HES7 | 1.148773 |
460 | 3321 | CDKN3 | 1.152745 |
461 | 3711 | LIMK1 | 1.153559 |
462 | 3503 | EGR1 | 1.156344 |
463 | 3701 | STK10 | 1.160598 |
464 | 3608 | MAP3K7IP1 | 1.161191 |
465 | 3730 | TESK1 | 1.162946 |
466 | 3156 | MSH2 | 1.163507 |
467 | 3571 | VAV1 | 1.164063 |
468 | 3668 | DAPK3 | 1.165365 |
469 | 3677 | HCK | 1.166105 |
470 | 3708 | RPS6KC1 | 1.166667 |
471 | 3110 | KIF13A | 1.167294 |
472 | 3185 | VCAM1 | 1.170254 |
473 | 3837 | PRKAA1 | 1.171443 |
474 | 3514 | HRAS | 1.171476 |
475 | 3371 | NM_006087 | 1.175311 |
476 | 3420 | NM_014109 | 1.176378 |
477 | 3669 | NTRK3 | 1.17801 |
478 | 2939 | TLK1 | 1.179137 |
479 | 3654 | VRK2 | 1.180868 |
480 | 3636 | STAT2 | 1.181562 |
481 | 3506 | XM_095827 | 1.18376 |
482 | 3728 | TIE | 1.184901 |
483 | 3496 | EIF4EBP2 | 1.188138 |
484 | 2994 | MATK | 1.188439 |
485 | 3353 | PGR | 1.188925 |
486 | 3771 | PIK3CA | 1.191131 |
487 | 3111 | KIF5B | 1.191167 |
488 | 3396 | HDAC10 | 1.192015 |
489 | 3330 | CDK9 | 1.194303 |
490 | 3705 | NM_012119 | 1.195395 |
491 | 3339 | CCNB2 | 1.195402 |
492 | 3005 | MERTK | 1.196303 |
493 | 3220 | ANAPC11 | 1.1994 |
494 | 3507 | NM_145754 | 1.199485 |
495 | 3418 | CENPA | 1.199564 |
496 | 3492 | PRKCQ | 1.199597 |
497 | 3499 | GRB2 | 1.204124 |
498 | 3667 | NM_016542 | 1.204923 |
499 | 3084 | KIF14 | 1.207333 |
500 | 3317 | CCNI | 1.208734 |
501 | 3457 | BAD | 1.2C8929 |
502 | 3819 | TACC3 | 1.209677 |
503 | 3377 | NM_006088 | 1.210588 |
504 | 3472 | MAP3K5 | 1.210677 |
505 | 2922 | NM_004783 | 1.211356 |
506 | 3453 | MAP2K2 | 1.212321 |
507 | 3724 | EPHA7 | 1.213738 |
508 | 3260 | STK11 | 1.214815 |
509 | 3675 | ADRBK1 | 1.215503 |
510 | 3379 | NM_032525 | 1.223512 |
511 | 2956 | PRKCL2 | 1.223938 |
512 | 3666 | PAK6 | 1.229403 |
513 | 3216 | CDC20 | 1.231173 |
514 | 3672 | SYK | 1.231714 |
515 | 3555 | RASA1 | 1.236402 |
516 | 3354 | RRM1 | 1.237695 |
517 | 3153 | RB1 | 1.237825 |
518 | 3253 | PRKCA | 1.239404 |
519 | 3146 | KIF21A | 1.240245 |
520 | 3756 | CDK5RAP2 | 1.242775 |
521 | 3721 | ANKRD3 | 1.245185 |
522 | 3224 | FBXO5 | 1.24973 |
523 | 3607 | TLE1 | 1.250329 |
524 | 2981 | ALK | 1.252514 |
525 | 2978 | AB067470 | 1.252713 |
526 | 3440 | FGFR3 | 1.253731 |
527 | 3578 | RREB1 | 1.256567 |
528 | 3393 | HDAC2 | 1.258824 |
529 | 3520 | NRAS | 1.263715 |
530 | 3190 | WNT4 | 1.265328 |
531 | 3463 | TEC | 1.265973 |
532 | 3621 | CTNNA2 | 1.26658 |
533 | 3425 | DLG7 | 1.267399 |
534 | 3311 | CDK10 | 1.269347 |
535 | 3567 | SHC1 | 1.270057 |
536 | 3753 | CDK5 | 1.276163 |
537 | 2989 | ACVR1B | 1.276215 |
538 | 3692 | AB007941 | 1.27931 |
539 | 3244 | PRKCZ | 1.279368 |
540 | 3092 | KIF12 | 1.279896 |
541 | 3487 | MAP2K3 | 1.280835 |
542 | 3813 | ANLN | 1.282313 |
543 | 3198 | ICAM1 | 1.285429 |
544 | 3697 | CAMK2G | 1.286036 |
545 | 3735 | PRKACB | 1.286694 |
546 | 3100 | GSK3B | 1.289078 |
547 | 3431 | NM_018454 | 1.289806 |
548 | 3615 | FZD2 | 1.292222 |
549 | 2947 | NM_007064 | 1.29381 |
550 | 3340 | CENPH | 1.293935 |
551 | 3172 | PLAU | 1.297571 |
552 | 3160 | TACSTD1 | 1.297585 |
553 | 3212 | NM_022662 | 1.301215 |
554 | 3098 | CENPE | 1.305802 |
555 | 3626 | DVL3 | 1.306682 |
556 | 3830 | NM_013296 | 1.307494 |
557 | 3713 | PRKG2 | 1.307933 |
558 | 3768 | ARAF1 | 1.308011 |
559 | 3493 | MAPK9 | 1.308449 |
560 | 3108 | K1F22 | 1.308726 |
561 | 3169 | NME1 | 1.310985 |
562 | 3125 | NM_031217 | 1.311267 |
563 | 3375 | AHCY | 1.311852 |
564 | 3583 | JUNB | 1.31241 |
565 | 3458 | ARAF1 | 1.315519 |
566 | 3612 | TCF1 | 1.316285 |
567 | 3294 | CCNF | 1.317748 |
568 | 3338 | CUL4A | 1.318527 |
569 | 3649 | CAMK2B | 1.322337 |
570 | 3576 | GRAP | 1.322985 |
571 | 3527 | DTX2 | 1.33023 |
572 | 3145 | C20orf23 | 1.334687 |
573 | 3180 | CD44 | 1.335574 |
574 | 3758 | RAD51L1 | 1.335901 |
575 | 3165 | FGF2 | 1.336082 |
576 | 3828 | KIF20A | 1.337004 |
577 | 3553 | CKS1B | 1.339383 |
578 | 3089 | KIFCI | 1.341566 |
579 | 3442 | ERBB4 | 1.345118 |
580 | 3554 | PIK3R3 | 1.347147 |
581 | 3613 | DVL1 | 1.347505 |
582 | 2985 | MKNK1 | 1.347934 |
583 | 3117 | ATM | 1.348967 |
584 | 3424 | EZH2 | 1.352941 |
585 | 3695 | PRKAA1 | 1.355145 |
586 | 3446 | PRKCG | 1.355556 |
587 | 3194 | RARB | 1.359932 |
588 | 3644 | EPHB1 | 1.36061 |
589 | 3700 | AB037782 | 1.361005 |
590 | 3599 | DTYMK | 1.361789 |
591 | 3729 | RYK | 1.361997 |
592 | 3114 | PIK3CD | 1.362808 |
593 | 3821 | ASPM | 1.363705 |
594 | 3373 | top2A | 1.363708 |
595 | 3563 | RAB3A | 1.365615 |
596 | 3764 | NOTCH4 | 1.36911 |
597 | 3628 | CTNNBL1 | 1.3702 |
598 | 3823 | NM_017779 | 1.372126 |
599 | 3715 | CDC42BPA | 1.372256 |
600 | 3562 | RASD1 | 1.372563 |
601 | 3784 | MAPK8 | 1.376577 |
602 | 3574 | SH3KBP1 | 1.384674 |
603 | 3594 | RAP2A | 1.393939 |
604 | 3662 | LCK | 1.3981 |
605 | 3787 | FZD4 | 1.399749 |
606 | 3316 | CCNA1 | 1.404295 |
607 | 3684 | STK38L | 1.406161 |
608 | 3610 | LEF1 | 1.407463 |
609 | 3390 | NM_080925 | 1.407563 |
610 | 3152 | APC | 1.414678 |
611 | 3149 | TP53 | 1.420044 |
612 | 3238 | MAP3K3 | 1.420428 |
613 | 3109 | KIF1C | 1.420608 |
614 | 3325 | CDKN1C | 1.42522 |
615 | 3314 | CCNG1 | 1.426516 |
616 | 3825 | NM_152562 | 1.428805 |
617 | 3588 | RHEB | 1.435039 |
618 | 3736 | PTK7 | 1.440171 |
619 | 3118 | NM_032559 | 1.440252 |
620 | 3521 | MET | 1.440418 |
621 | 3096 | AKT1 | 1.440951 |
622 | 3361 | IMPDH2 | 1.442308 |
623 | 3582 | GRAP2 | 1.444349 |
624 | 3584 | RASAL2 | 1.450119 |
625 | 3801 | PSEN1 | 1.466292 |
626 | 3803 | MPHOSPH1 | 1.470276 |
627 | 2938 | ALS2CR7 | 1.471357 |
628 | 3106 | KIF9 | 1.47493 |
629 | 3313 | CCNG2 | 1.48267 |
630 | 3792 | ARHGEF1 | 1.48329 |
631 | 3210 | NM_013366 | 1.48366 |
632 | 3820 | NM_018410 | 1.483709 |
633 | 2932 | MAPKAPK3 | 1.488 |
634 | 3747 | GSK3A | 1.491773 |
635 | 2962 | MAP4K2 | 1.495448 |
636 | 3699 | NM_032844 | 1.502812 |
637 | 3189 | MYB | 1.504618 |
638 | 3629 | AXIN1 | 1.505556 |
639 | 2941 | DYRK3 | 1.505717 |
640 | 3818 | A1338451 | 1.511194 |
641 | 2919 | OSR1 | 1.512906 |
642 | 3140 | XM_089006 | 1.518548 |
643 | 3229 | PRKCL1 | 1.525203 |
644 | 3510 | CDK4 | 1.529837 |
645 | 3319 | CCND1 | 1.531034 |
646 | 3159 | RET | 1.536506 |
647 | 3242 | PRKCB1 | 1.540024 |
648 | 3519 | NTRK1 | 1.547773 |
649 | 3808 | CKAP2 | 1.554545 |
650 | 2988 | FRK | 1.557214 |
651 | 2944 | MARK1 | 1.557763 |
652 | 2971 | DAPK2 | 1.55938 |
653 | 3299 | CUL1 | 1.560841 |
654 | 3660 | DMPK | 1.5625 |
655 | 3515 | PDGFRB | 1.562977 |
656 | 3522 | KRAS2 | 1.564353 |
657 | 3004 | MAP3K1 | 1.570175 |
658 | 3395 | HDAC5 | 1.571159 |
659 | 3468 | ABL2 | 1.571225 |
660 | 3529 | DTX1 | 1.57276 |
661 | 3329 | CDC42 | 1.580386 |
662 | 3704 | ACVR2B | 1.58046 |
663 | 3827 | NM_018123 | 1.581315 |
664 | 3456 | FLT1 | 1.583826 |
665 | 3310 | CDC34 | 1.585818 |
666 | 3331 | CDC25B | 1.585938 |
667 | 3368 | top3A | 1.58728 |
668 | 3126 | KIF3B | 1.588728 |
669 | 3780 | MCM3 | 1.590296 |
670 | 3128 | AKT2 | 1.592696 |
671 | 3598 | PCNA | 1.59319 |
672 | 3535 | SKP2 | 1.593333 |
673 | 2955 | PAK1 | 1.59552 |
674 | 3234 | CDK2 | 1.596033 |
675 | 3138 | KIF17 | 1.604846 |
676 | 3632 | WISP1 | 1.607319 |
677 | 3611 | CTNNAL1 | 1.611386 |
678 | 3300 | CDC14B | 1.611486 |
679 | 3511 | XM_168069 | 1.614698 |
680 | 3144 | KIF4B | 1.619674 |
681 | 3627 | CTNNA1 | 1.620915 |
682 | 3337 | CDKN1A | 1.626582 |
683 | 3202 | MCC | 1.627957 |
684 | 3143 | NM_017596 | 1.628521 |
685 | 3186 | ETS1 | 1.635593 |
686 | 3432 | PRC1 | 1.637647 |
687 | 3556 | RAP1A | 1.638173 |
688 | 3335 | CDK5R2 | 1.656172 |
689 | 2933 | MAP4K5 | 1.656522 |
690 | 2927 | MAPK13 | 1.659401 |
691 | 2973 | NEK1 | 1.664311 |
692 | 3538 | NFKB2 | 1.667808 |
693 | 3602 | MCM3 | 1.678819 |
694 | 3603 | POLS | 1.678937 |
695 | 3630 | WISP3 | 1.679045 |
696 | 3447 | PRKCM | 1.680152 |
697 | 3402 | HDAC4 | 1.68123 |
698 | 3133 | XM_168069 | 1.681935 |
699 | 3428 | ECT2 | 1.690096 |
700 | 3720 | AB002301 | 1.691718 |
701 | 3793 | MAPRE1 | 1.693966 |
702 | 3681 | SRPK1 | 1.700611 |
703 | 3817 | NM_019013 | 1.702326 |
704 | 3136 | XM_066649 | 1.708388 |
705 | 3355 | GUK1 | 1.710938 |
706 | 3087 | PTEN | 1.716866 |
707 | 3579 | PDZGEF2 | 1.717714 |
708 | 3168 | DCC | 1.719083 |
709 | 3151 | MLH1 | 1.72077 |
710 | 3217 | NM_014885 | 1.722045 |
711 | 3191 | WNT2 | 1.728016 |
712 | 3765 | CREBBP | 1.72973 |
713 | 3655 | CAMK2D | 1.733773 |
714 | 3407 | HDAC9 | 1.748784 |
715 | 3255 | CDK7 | 1.75 |
716 | 3295 | CDK2AP1 | 1.75 |
717 | 3192 | WNT1 | 1.751208 |
718 | 3333 | CCNT2 | 1.761104 |
719 | 3703 | AK024504 | 1.764425 |
720 | 3760 | CDKL5 | 1.769444 |
721 | 2948 | MYO3A | 1.769759 |
722 | 3800 | CHFR | 1.772809 |
723 | 3544 | IRS1 | 1.776668 |
724 | 3235 | CHEK1 | 1.776886 |
725 | 3137 | KIF26A | 1.782366 |
726 | 3673 | DDR1 | 1.792507 |
727 | 3336 | CDC37 | 1.807985 |
728 | 3725 | EPHA4 | 1.820076 |
729 | 3404 | PPARG | 1.822581 |
730 | 3604 | TK2 | 1.82846 |
731 | 3738 | PRKWNK3 | 1.836245 |
732 | 3141 | NM_145754 | 1.843889 |
733 | 3451 | MAP3K4 | 1.855556 |
734 | 3417 | HDAC3 | 1.857143 |
735 | 3508 | KIF25 | 1.871592 |
736 | 3575 | LATS2 | 1.879574 |
737 | 3761 | WT1 | 1.88089 |
738 | 3723 | NM_018401 | 1.88722 |
739 | 3719 | BMPR2 | 1.890545 |
740 | 3204 | CDC16 | 1.892826 |
741 | 3467 | MAP2K7 | 1.894459 |
742 | 2986 | ACVR2 | 1.896882 |
743 | 3218 | CDC23 | 1.904255 |
744 | 3791 | NM_005200 | 1.913043 |
745 | 3804 | NM_024322 | 1.920139 |
746 | 3558 | RALA | 1.92029 |
747 | 3824 | MAPRE3 | 1.940871 |
748 | 3622 | FZD9 | 1.988166 |
749 | 3205 | NM_139286 | 1.997054 |
750 | 3221 | top3B | 1.997534 |
751 | 3794 | WASL | 1.998403 |
752 | 3637 | STAT4 | 2.005199 |
753 | 3834 | CHEK1 | 2.01625 |
754 | 3400 | BCL2 | 2.045028 |
755 | 3223 | NM_016263 | 2.045139 |
756 | 3358 | top2B | 2.050562 |
757 | 3512 | TGFBR1 | 2.062016 |
758 | 3259 | MAPK8 | 2.064081 |
759 | 3742 | RHOK | 2.075949 |
760 | 2946 | NM_017719 | 2.078131 |
761 | 3406 | TERT | 2.10274 |
762 | 3206 | ANAPC5 | 2.159615 |
763 | 3531 | NM_021170 | 2.163086 |
764 | 3008 | SGK2 | 2.1766 |
765 | 3706 | C20orf97 | 2.1875 |
766 | 3254 | CSF1R | 2.196822 |
767 | 3439 | EGR2 | 2.213333 |
768 | 2970 | AATK | 2.235211 |
769 | 3528 | TCF3 | 2.273649 |
770 | 3327 | CDC45L | 2.288265 |
771 | 3551 | STAT3 | 2.29125 |
772 | 3001 | PRKY | 2.313131 |
773 | 3734 | BMPR1B | 2.330839 |
774 | 3095 | KIF2C | 2.336785 |
775 | 3222 | PTTGI | 2.347826 |
776 | 3532 | NM_019089 | 2.352437 |
777 | 3547 | FOXO1A | 2.352444 |
778 | 3671 | STK4 | 2.362408 |
779 | 3781 | SRC | 2.37859 |
780 | 3789 | ELKI | 2.394828 |
781 | 3247 | NM_018492 | 2.480851 |
782 | 3586 | RASA2 | 2.506796 |
783 | 3727 | GPRK6 | 2.553987 |
784 | 3689 | BLK | 2.584588 |
785 | 3777 | ABL1 | 2.615226 |
786 | 3399 | HSPCB | 2.632207 |
787 | 2958 | PRKACA | 2.635514 |
788 | 3304 | CCNE2 | 2.677656 |
789 | 3617 | CTNNBIP1 | 2.698292 |
790 | 3225 | NM_013367 | 2.714286 |
791 | 3619 | FRAT1 | 2.728111 |
792 | 3121 | PIK3C2A | 2.828125 |
793 | 3816 | NM_017769 | 2.847273 |
794 | 3134 | XM_170783 | 2.923286 |
795 | 3737 | NM_016457 | 2.940451 |
796 | 3135 | XM_064050 | 3.063002 |
797 | 3129 | STK6 | 3.146434 |
798 | 3564 | RALBP1 | 3.170605 |
799 | 3580 | ELK1 | 3.356401 |
800 | 3157 | NF1 | 3.402273 |
801 | 3638 | STAT5A | 3.754386 |
802 | 3241 | WEE1 | 3.801887 |
803 | 3718 | PTK6 | 4.317857 |
804 | 3712 | RPS6KA6 | 5.356624 |
805 | 3158 | BRCA1 | 5.821429 |
806 | 3642 | EPHB3 | 6.43 |
807 | 3150 | BRCA2 | 14.13136 |
The average sensitization multiple that Table II C Zorubicin causes
Gene I | BiolD | Gene | The mean value of three |
1 | 2514 | PLK | 0.094489 |
2 | 3099 | PLK | 0.195626 |
3 | 3099 | PLK | 0.211482 |
4 | 3099 | PLK | 0.211747 |
5 | 3099 | PLK | 0.219626 |
6 | 3099 | PLK | 0.227603 |
7 | 3099 | PLK | 0.235482 |
8 | 3099 | PLK | 0.235683 |
9 | 3099 | PLK | 0.235747 |
10 | 3099 | PLK | 0.251539 |
11 | 3099 | PLK | 0.251603 |
12 | 3099 | PLK | 0.259683 |
13 | 3099 | PLK | 0.275539 |
14 | 3099 | PLK | 0.282503 |
15 | 3099 | PLK | 0.298359 |
16 | 3099 | PLK | 0.298624 |
17 | 3099 | PLK | 0.31448 |
18 | 3099 | PLK | 0.32256 |
19 | 3534 | PLK | 0.330807 |
20 | 3099 | PLK | 0.338416 |
21 | 3099 | PLK | 0.395491 |
22 | 3099 | PLK | 0.411612 |
23 | 3006 | PLK | 0.415454 |
24 | 3099 | PLK | 0.419491 |
25 | 3099 | PLK | 0.435548 |
26 | 3099 | PLK | 0.435612 |
27 | 3433 | PLK | 0.435845 |
28 | 3391 | PLK | 0.440842 |
29 | 3099 | PLK | 0.459548 |
30 | 3099 | PLK | 0.482368 |
31 | 3099 | PLK | 0.498489 |
32 | 3322 | CCNA2 | 0.512614 |
33 | 3099 | PLK | 0.522425 |
34 | 3805 | C20orf1 | 0.562328 |
35 | 3423 | 0.613084 | |
36 | 3600 | RRM2B | 0.659243 |
37 | 3305 | CDC2L5 | 0.68014 |
38 | 3542 | PPP2CA | 0.695506 |
39 | 3266 | PLK | 0.696721 |
40 | 3228 | CDC27 | 0.70157 |
41 | 3464 | INSR | 0.70706 |
42 | 3326 | CCT7 | 0.724986 |
43 | 3740 | STK35 | 0.754807 |
44 | 3731 | CSNK1E | 0.765738 |
45 | 3416 | IKBKE | 0.773235 |
46 | 3293 | CDC25A | 0.77957 |
47 | 3309 | CCND2 | 0.791487 |
48 | 3350 | ADA | 0.800034 |
49 | 3812 | CDCA8 | 0.815766 |
50 | 3354 | RRM1 | 0.817751 |
51 | 3446 | PRKCG | 0.822809 |
52 | 3648 | CLK1 | 0.824307 |
53 | 3509 | KIF21A | 0.826427 |
54 | 3526 | HES6 | 0.826991 |
55 | 3250 | CHEK2 | 0.828202 |
56 | 3262 | LATS1 | 0.82944 |
57 | 3359 | TUBA1 | 0.839308 |
58 | 3344 | CDKL1 | 0.840425 |
59 | 2984 | EPHB6 | 0.846685 |
60 | 3702 | MAP3K13 | 0.84685 |
61 | 3838 | PRKAA2 | 0.853115 |
62 | 3422 | SMC4L1 | 0.854651 |
63 | 3332 | CDC5L | 0.85491 |
64 | 3750 | CAMK2A | 0.857171 |
65 | 3686 | MAP3K8 | 0.8599 |
66 | 3226 | RBX1 | 0.862335 |
67 | 3438 | DTR | 0.863218 |
68 | 3318 | CUL2 | 0.863485 |
69 | 3454 | FLT4 | 0.864511 |
70 | 3366 | TYMS | 0.866092 |
71 | 3444 | NEK3 | 0.866318 |
72 | 3397 | BUB3 | 0.867363 |
73 | 3007 | MAP3K14 | 0.86906 |
74 | 3373 | top2A | 0.875387 |
75 | 2934 | IRAK2 | 0.875671 |
76 | 3188 | PMS2 | 0.876644 |
77 | 3461 | KIT | 0.876727 |
78 | 3398 | HDAC11 | 0.878587 |
79 | 3665 | PAK4 | 0.879213 |
80 | 3494 | MAPK4 | 0.879947 |
81 | 3303 | CDKN2D | 0.88429 |
82 | 2925 | FYN | 0.885569 |
83 | 3437 | FGFR1 | 0.889075 |
84 | 3219 | CENPC1 | 0.889832 |
85 | 3491 | PRKCE | 0.891708 |
86 | 3105 | BUB1 | 0.892262 |
87 | 3609 | FZD3 | 0.89297 |
88 | 3421 | ORC6L | 0.893859 |
89 | 3414 | HDAC7A | 0.894925 |
90 | 3342 | CCNT1 | 0.89645 |
91 | 3193 | FGF3 | 0.897275 |
92 | 3203 | ITGA5 | 0.89915 |
93 | 3679 | CLK2 | 0.899792 |
94 | 3656 | PRKCN | 0.903305 |
95 | 3677 | HCK | 0.903727 |
96 | 3172 | PLAU | 0.904045 |
97 | 2999 | FES | 0.904351 |
98 | 3161 | WT1 | 0.907863 |
99 | 3230 | MAP2K1 | 0.908157 |
100 | 2937 | 0.910875 | |
101 | 3502 | PRKCH | 0.913184 |
102 | 3317 | CCN1 | 0.913695 |
103 | 3086 | KIF11 | 0.914508 |
104 | 3412 | KIF25 | 0.915671 |
105 | 3710 | GPRK2L | 0.917359 |
106 | 3585 | GAB1 | 0.91762 |
107 | 3807 | SPAG5 | 0.918025 |
108 | 3815 | MAPRE2 | 0.919461 |
109 | 3646 | 0.920311 | |
110 | 3000 | BMX | 0.920926 |
111 | 3365 | PRIM1 | 0.922943 |
112 | 3574 | SH3KBP1 | 0.924261 |
113 | 3485 | DHX8 | 0.924589 |
114 | 3527 | DTX2 | 0.92511 |
115 | 3378 | 0.927814 | |
116 | 3799 | ACTR3 | 0.929286 |
117 | 3822 | GTSE1 | 0.929871 |
118 | 3100 | GSK3B | 0.932676 |
119 | 3206 | ANAPC5 | 0.932816 |
120 | 3351 | ESR1 | 0.932858 |
121 | 3623 | CTNND1 | 0.932974 |
122 | 3601 | POLE | 0.935664 |
123 | 3097 | KIF20A | 0.939338 |
124 | 2991 | NPR1 | 0.941392 |
125 | 2926 | 0.943073 | |
126 | 3717 | NTRK2 | 0.94323 |
127 | 3162 | MADH2 | 0.953335 |
128 | 3783 | KRAS2 | 0.954957 |
129 | 3660 | DMPK | 0.955308 |
130 | 3236 | PTK2B | 0.955874 |
131 | 3088 | KIF13B | 0.960206 |
132 | 3774 | CDC45L | 0.961565 |
133 | 3540 | PPP2CB | 0.96255 |
134 | 3251 | ABL1 | 0.96267 |
135 | 3498 | CSNK1A1 | 0.963185 |
136 | 3307 | CDC6 | 0.963749 |
137 | 3830 | 0.96419 | |
138 | 3374 | POLR2A | 0.964327 |
139 | 3413 | KIF23 | 0.967774 |
140 | 3296 | CDKN2C | 0.967818 |
141 | 3132 | KIF23 | 0.96794 |
142 | 3708 | RPS6KC1 | 0.969675 |
143 | 3445 | BMPR1A | 0.970178 |
144 | 3694 | STK38 | 0.970842 |
145 | 3566 | JUN | 0.971389 |
146 | 3140 | 0.97186 | |
147 | 3571 | VAV1 | 0.972374 |
148 | 2993 | SRMS | 0.972957 |
149 | 3268 | CDC25C | 0.973198 |
150 | 3835 | CHEK2 | 0.973353 |
151 | 3557 | JUND | 0.973868 |
152 | 3195 | CDH1 | 0.973895 |
153 | 3375 | AHCY | 0.974215 |
154 | 3163 | THRA | 0.976052 |
155 | 3164 | MYCL1 | 0.979364 |
156 | 3798 | ACTR2 | 0.980521 |
157 | 3392 | BIRC5 | 0.980792 |
158 | 3196 | ARHI | 0.980973 |
159 | 3536 | PIK3C3 | 0.981403 |
160 | 2950 | NEK6 | 0.981709 |
161 | 3773 | WNT2 | 0.982648 |
162 | 3776 | NOTCH2 | 0.983584 |
163 | 3814 | HMMR | 0.983597 |
164 | 3234 | CDK2 | 0.983724 |
165 | 2982 | CDC2L2 | 0.984121 |
166 | 3826 | 0.985249 | |
167 | 2953 | MAPK11 | 0.987788 |
168 | 3403 | AURKC | 0.988679 |
169 | 3586 | RASA2 | 0.989648 |
170 | 3503 | EGR1 | 0.991443 |
171 | 3166 | PMS1 | 0.99314 |
172 | 3394 | HDAC6 | 0.994139 |
173 | 3652 | PDGFRA | 0.994658 |
174 | 3625 | CTBP2 | 0.994928 |
175 | 3294 | CCNF | 0.995133 |
176 | 3260 | STK11 | 0.998488 |
177 | 2968 | STK17B | 0.998826 |
178 | 3703 | 0.999818 | |
179 | 3577 | RAB2L | 1.00021 |
180 | 3184 | TSG101 | 1.00109 |
181 | 2927 | MAPK13 | 1.001159 |
182 | 3116 | KIF5A | 1.002239 |
183 | 3496 | EIF4EBP2 | 1.005451 |
184 | 3741 | RPS6KB2 | 1.00589 |
185 | 3298 | CCNE1 | 1.005922 |
186 | 2990 | 1.006496 | |
187 | 3142 | KIF25 | 1.006522 |
188 | 3218 | CDC23 | 1.009586 |
189 | 3517 | MYC | 1.010689 |
190 | 2997 | MST1R | 1.011122 |
191 | 3003 | FER | 1.012506 |
192 | 3700 | 1.013542 | |
193 | 3470 | RPS6KA1 | 1.013802 |
194 | 3439 | EGR2 | 1.013847 |
195 | 3429 | MCM6 | 1.014653 |
196 | 3372 | TUBA8 | 1.017048 |
197 | 3556 | RAP1A | 1.017133 |
198 | 3155 | ATR | 1.017435 |
199 | 3649 | CAMK2B | 1.017461 |
200 | 3501 | RPS6KA2 | 1.018616 |
201 | 3336 | CDC37 | 1.019161 |
202 | 2928 | IRAK1 | 1.021732 |
203 | 3733 | MYLK2 | 1.021742 |
204 | 2960 | LYN | 1.022112 |
205 | 3301 | CCNB1 | 1.022891 |
206 | 3743 | RAGE | 1.023372 |
207 | 3525 | NOTCH4 | 1.02341 |
208 | 3767 | FZD3 | 1.023646 |
209 | 2954 | ROCK2 | 1.02397 |
210 | 3475 | EPHB4 | 1.024709 |
211 | 3635 | STAT1 | 1.026128 |
212 | 3746 | HUNK | 1.026176 |
213 | 2977 | RIPK3 | 1.0272 |
214 | 3573 | 1.028343 | |
215 | 3751 | BCR | 1.028418 |
216 | 3112 | KNSL7 | 1.029109 |
217 | 3488 | AURKB | 1.029885 |
218 | 3356 | TUBG1 | 1.029908 |
219 | 3364 | HPRT1 | 1.030247 |
220 | 3465 | EPHA1 | 1.032043 |
221 | 3828 | KIF20A | 1.032108 |
222 | 3434 | DDX6 | 1.03425 |
223 | 3143 | 1.03439 | |
224 | 3212 | 1.034473 | |
225 | 3725 | EPHA4 | 1.034871 |
226 | 3473 | DAPK1 | 1.035466 |
227 | 3581 | RASGRP1 | 1.036407 |
228 | 3357 | PRIM2A | 1.036773 |
229 | 3469 | AAK1 | 1.037538 |
230 | 3171 | VHL | 1.038422 |
231 | 3123 | AKT3 | 1.039278 |
232 | 3572 | RASA3 | 1.04084 |
233 | 3615 | FZD2 | 1.042378 |
234 | 3658 | STK18 | 1.043083 |
235 | 3261 | ERBB2 | 1.044345 |
236 | 3220 | ANAPC11 | 1.0449 |
237 | 3639 | STAT6 | 1.045395 |
238 | 2959 | PIM2 | 1.048207 |
239 | 2935 | MAPK6 | 1.050943 |
240 | 3752 | CCNB3 | 1.051148 |
241 | 3431 | 1.05315 | |
242 | 3101 | MAPK14 | 1.054104 |
243 | 3462 | TGFBR2 | 1.056272 |
244 | 3319 | CCND1 | 1.057299 |
245 | 3592 | SOS2 | 1.058842 |
246 | 3655 | CAMK2D | 1.061571 |
247 | 3513 | ROS1 | 1.062804 |
248 | 3297 | CCT2 | 1.064889 |
249 | 3549 | PIK3R2 | 1.066314 |
250 | 2998 | MAPK7 | 1.066798 |
251 | 3334 | CUL4B | 1.066807 |
252 | 3381 | POLR2B | 1.068615 |
253 | 3633 | CTBP1 | 1.069269 |
254 | 3678 | PFTK1 | 1.07042 |
255 | 2987 | RNASEL | 1.072118 |
256 | 3256 | CDC2L1 | 1.073967 |
257 | 3558 | RALA | 1.074961 |
258 | 3749 | 1.075156 | |
259 | 3252 | NEK2 | 1.075822 |
260 | 2919 | OSR1 | 1.077885 |
261 | 3393 | HDAC2 | 1.077906 |
262 | 3747 | GSK3A | 1.078401 |
263 | 3410 | RPS27 | 1.078517 |
264 | 3107 | KIF2 | 1.078686 |
265 | 3654 | VRK2 | 1.081195 |
266 | 3533 | HES7 | 1.081287 |
267 | 2983 | GUCY2C | 1.083605 |
268 | 3555 | RASA1 | 1.084083 |
269 | 3258 | MOS | 1.084874 |
270 | 3180 | CD44 | 1.085294 |
271 | 3124 | KIF4A | 1.086165 |
272 | 3179 | PDGFB | 1.086599 |
273 | 3209 | MAD2L2 | 1.088835 |
274 | 3295 | CDK2AP1 | 1.089703 |
275 | 3726 | MAPKAPK2 | 1.09032 |
276 | 3674 | CSNK1D | 1.090974 |
277 | 3616 | FZD1 | 1.091935 |
278 | 3452 | JAK1 | 1.092015 |
279 | 3823 | 1.092187 | |
280 | 3745 | CAMKK2 | 1.092307 |
281 | 3149 | TP53 | 1.092755 |
282 | 3561 | FOS | 1.092859 |
283 | 3836 | TP53 | 1.093641 |
284 | 3170 | BLM | 1.094952 |
285 | 2930 | ITK | 1.095322 |
286 | 3744 | PRKWNK4 | 1.095854 |
287 | 3401 | HDAC1 | 1.096531 |
288 | 3300 | CDC14B | 1.096651 |
289 | 3348 | DCK | 1.096689 |
290 | 3405 | HDAC8 | 1.096956 |
291 | 3239 | CDK6 | 1.097696 |
292 | 3640 | STAT5B | 1.098035 |
293 | 2992 | PRKR | 1.098133 |
294 | 3548 | RAC1 | 1.09835 |
295 | 3306 | CDC14A | 1.098874 |
296 | 2943 | DYRK2 | 1.099617 |
297 | 3127 | MAPK1 | 1.102044 |
298 | 3716 | ULK1 | 1.104258 |
299 | 2922 | 1.106102 | |
300 | 3160 | TACSTD1 | 1.107397 |
301 | 2964 | RIPK2 | 1.109482 |
302 | 3634 | BTRC | 1.110007 |
303 | 3576 | GRAP | 1.110227 |
304 | 3833 | ATR | 1.110614 |
305 | 3837 | PRKAA1 | 1.111073 |
306 | 2939 | TLK1 | 1.111429 |
307 | 3125 | 1.11143 | |
308 | 3299 | CUL1 | 1.111864 |
309 | 3813 | ANLN | 1.112297 |
310 | 3756 | CDK5RAP2 | 1.112508 |
311 | 2976 | NEK7 | 1.112602 |
312 | 2965 | 1.11309 | |
313 | 3784 | MAPK8 | 1.114132 |
314 | 3653 | NPR2 | 1.115282 |
315 | 3302 | CDK8 | 1.115429 |
316 | 3628 | CTNNBL1 | 1.115905 |
317 | 3664 | STK17A | 1.115938 |
318 | 3504 | PIK3CB | 1.117357 |
319 | 3395 | HDAC5 | 1.118952 |
320 | 3369 | 1.119457 | |
321 | 3243 | 1.119572 | |
322 | 3715 | CDC42BPA | 1.119975 |
323 | 2924 | STK25 | 1.122952 |
324 | 3568 | PLD1 | 1.123878 |
325 | 3676 | MAP4K1 | 1.124218 |
326 | 3343 | CENPJ | 1.127564 |
327 | 3238 | MAP3K3 | 1.127647 |
328 | 3424 | EZH2 | 1.127778 |
329 | 3418 | CENPA | 1.128399 |
330 | 3829 | KIF2C | 1.128457 |
331 | 3476 | PRKCD | 1.128572 |
332 | 3407 | HDAC9 | 1.129023 |
333 | 2951 | 1.13065 | |
334 | 3685 | LTK | 1.130723 |
335 | 2942 | TTN | 1.131132 |
336 | 3085 | KIF5C | 1.133235 |
337 | 3367 | DHFR | 1.133721 |
338 | 3362 | NR3C1 | 1.134725 |
339 | 3400 | BCL2 | 1.134785 |
340 | 3800 | CHFR | 1.134967 |
341 | 3103 | PIK3CA | 1.135082 |
342 | 3711 | LIMK1 | 1.135687 |
343 | 3165 | FGF2 | 1.136323 |
344 | 3213 | 1.137328 | |
345 | 3370 | AR | 1.137843 |
346 | 3772 | RPS6KB1 | 1.138023 |
347 | 3189 | MYB | 1.138695 |
348 | 3631 | WISP2 | 1.138989 |
349 | 2945 | CDC42BPB | 1.140434 |
350 | 3593 | VAV2 | 1.141048 |
351 | 3338 | CUL4A | 1.141509 |
352 | 3092 | K1F12 | 1.14183 |
353 | 3782 | SOS1 | 1.14272 |
354 | 2989 | ACVR1B | 1.143948 |
355 | 3808 | CKAP2 | 1.144074 |
356 | 3310 | CDC34 | 1.14429 |
357 | 3760 | CDKL5 | 1.144621 |
358 | 3159 | RET | 1.144761 |
359 | 3508 | KIF25 | 1.144865 |
360 | 3788 | PRKCE | 1.145993 |
361 | 3231 | ILK | 1.146387 |
362 | 3471 | MAPK10 | 1.146497 |
363 | 3668 | DAPK3 | 1.14781 |
364 | 3595 | FEN1 | 1.14853 |
365 | 3775 | NOTCH1 | 1.150372 |
366 | 3145 | C20orf23 | 1.151785 |
367 | 3570 | RALGDS | 1.152146 |
368 | 2972 | KSR | 1.152379 |
369 | 3441 | PRKCI | 1.152901 |
370 | 3737 | 1.153373 | |
371 | 3463 | TEC | 1.154814 |
372 | 3748 | 1.155977 | |
373 | 3816 | 1.156751 | |
374 | 3582 | GRAP2 | 1.158058 |
375 | 3360 | RRM2 | 1.158514 |
376 | 3516 | ARHA | 1.15962 |
377 | 3312 | CUL3 | 1.160258 |
378 | 3005 | MERTK | 1.160604 |
379 | 3456 | FLT1 | 1.160651 |
380 | 3567 | SHC1 | 1.161312 |
381 | 3647 | YES1 | 1.161861 |
382 | 3447 | PRKCM | 1.162427 |
383 | 3739 | 1.163543 | |
384 | 3181 | ST5 | 1.163581 |
385 | 3466 | JAK3 | 1.164099 |
386 | 3311 | CDK10 | 1.1651 |
387 | 3486 | RPS6KA3 | 1.165517 |
388 | 3779 | CTNNA1 | 1.165697 |
389 | 3148 | LIG1 | 1.166358 |
390 | 3683 | 1.167226 | |
391 | 3544 | IRS1 | 1.167527 |
392 | 3335 | CDK5R2 | 1.167989 |
393 | 3821 | ASPM | 1.167998 |
394 | 3108 | KIF22 | 1.168525 |
395 | 3168 | DCC | 1.170395 |
396 | 3182 | MYCN | 1.172038 |
397 | 3119 | CDKN1B | 1.172505 |
398 | 3692 | 1.173629 | |
399 | 3687 | CAMK4 | 1.17436 |
400 | 3420 | 1.175153 | |
401 | 3762 | MCC | 1.175576 |
402 | 3519 | NTRK1 | 1.175989 |
403 | 3257 | IGF1R | 1.176551 |
404 | 3769 | PLAU | 1.176774 |
405 | 3339 | CCNB2 | 1.177549 |
406 | 3682 | DYRK1A | 1.178203 |
407 | 3240 | MAPK12 | 1.178713 |
408 | 3156 | MSH2 | 1.17907 |
409 | 2936 | SGKL | 1.17989 |
410 | 2920 | EIF2AK3 | 1.179969 |
411 | 3670 | MAP3K10 | 1.180357 |
412 | 3207 | MAD1L1 | 1.181963 |
413 | 3630 | WISP3 | 1.182009 |
414 | 3153 | RB1 | 1.183084 |
415 | 3632 | WISP1 | 1.183165 |
416 | 3824 | MAPRE3 | 1.183387 |
417 | 3624 | CTNNB1 | 1.18419 |
418 | 3151 | MLH1 | 1.185254 |
419 | 3495 | FGFR2 | 1.185537 |
420 | 3349 | IMPDH1 | 1.185827 |
421 | 2932 | MAPKAPK3 | 1.186058 |
422 | 3130 | FRAP1 | 1.186158 |
423 | 3714 | 1.188036 | |
424 | 3467 | MAP2K7 | 1.188179 |
425 | 3727 | GPRK6 | 1.188457 |
426 | 3500 | SGK | 1.189014 |
427 | 3638 | STAT5A | 1.189492 |
428 | 3242 | PRKCB1 | 1.191673 |
429 | 3588 | RHEB | 1.194214 |
430 | 2940 | DCAMKL1 | 1.194443 |
431 | 3222 | PTTG1 | 1.194583 |
432 | 3411 | HIF1A | 1.194933 |
433 | 2952 | PRKG1 | 1.197336 |
434 | 3539 | PLCG2 | 1.198326 |
435 | 3797 | ARHGEF9 | 1.20036 |
436 | 2969 | 1.201116 | |
437 | 3194 | RARB | 1.201145 |
438 | 3490 | ERBB3 | 1.202371 |
439 | 3197 | ARHB | 1.2033 |
440 | 3347 | top1 | 1.203483 |
441 | 2966 | 1.203678 | |
442 | 3089 | KIFC1 | 1.204418 |
443 | 3232 | KDR | 1.205127 |
444 | 3090 | KIF3C | 1.205488 |
445 | 3599 | DTYMK | 1.205645 |
446 | 3139 | 1.206674 | |
447 | 3695 | PRKAA1 | 1.20878 |
448 | 3425 | DLG7 | 1.209098 |
449 | 3535 | SKP2 | 1.20949 |
450 | 3327 | CDC45L | 1.209854 |
451 | 3651 | VRK1 | 1.210029 |
452 | 3569 | RASGRP2 | 1.210373 |
453 | 3246 | RPS6KB1 | 1.210471 |
454 | 3131 | KIF1B | 1.21146 |
455 | 3671 | STK4 | 1.212033 |
456 | 3757 | CLK4 | 1.212447 |
457 | 2985 | MKNK1 | 1.212627 |
458 | 2988 | FRK | 1.213049 |
459 | 3432 | PRC1 | 1.2136 |
460 | 3699 | 1.214212 | |
461 | 3008 | SGK2 | 1.21451 |
462 | 2996 | MAPK3 | 1.217258 |
463 | 3399 | HSPCB | 1.217278 |
464 | 3610 | LEF1 | 1.219128 |
465 | 2980 | RIPK1 | 1.220712 |
466 | 3675 | ADRBK1 | 1.22227 |
467 | 3663 | ALS2CR2 | 1.223782 |
468 | 3468 | ABL2 | 1.223785 |
469 | 2961 | PIM1 | 1.223874 |
470 | 3804 | 1.224331 | |
471 | 3594 | RAP2A | 1.226644 |
472 | 3377 | 1.227759 | |
473 | 3341 | APLP2 | 1.229895 |
474 | 3524 | NOTCH3 | 1.230078 |
475 | 3253 | PRKCA | 1.233866 |
476 | 3518 | NFKB1 | 1.23456 |
477 | 3328 | CCNC | 1.236473 |
478 | 3563 | RAB3A | 1.237081 |
479 | 3765 | CREBBP | 1.23722 |
480 | 2979 | PAK2 | 1.237809 |
481 | 3235 | CHEK1 | 1.239472 |
482 | 3146 | KIF21A | 1.239694 |
483 | 3340 | CENPH | 1.239979 |
484 | 3215 | MAD2L1 | 1.24524 |
485 | 3379 | 1.245359 | |
486 | 3662 | LCK | 1.245594 |
487 | 3754 | CDKL2 | 1.247567 |
488 | 3187 | WNT7B | 1.247786 |
489 | 3552 | PDK1 | 1.248615 |
490 | 3618 | DVL2 | 1.249105 |
491 | 3602 | MCM3 | 1.249136 |
492 | 3564 | RALBP1 | 1.249919 |
493 | 3404 | PPARG | 1.252208 |
494 | 3248 | JAK2 | 1.252557 |
495 | 3147 | CDKN2A | 1.252718 |
496 | 3358 | top2B | 1.253573 |
497 | 3459 | EGFR | 1.255853 |
498 | 3249 | MAP2K6 | 1.256087 |
499 | 3254 | CSF1R | 1.258457 |
500 | 2949 | MYO3B | 1.259934 |
501 | 3157 | NF1 | 1.260606 |
502 | 3680 | CLK3 | 1.262403 |
503 | 3113 | CNK | 1.262742 |
504 | 3825 | 1.263089 | |
505 | 3667 | 1.26407 | |
506 | 3753 | CDK5 | 1.264077 |
507 | 3553 | CKS1B | 1.265301 |
508 | 2933 | MAP4K5 | 1.2656S5 |
509 | 3796 | ARHGEF6 | 1.265751 |
510 | 3419 | RFC4 | 1.266922 |
511 | 3460 | CSK | 1.266969 |
512 | 3094 | KIF3A | 1.26728 |
513 | 3736 | PTK7 | 1.267303 |
514 | 3707 | TXK | 1.268516 |
515 | 3791 | 1.26868 | |
516 | 3523 | NOTCH2 | 1.26965 |
517 | 3755 | CDKL3 | 1.271644 |
518 | 3204 | CDC16 | 1.271646 |
519 | 3353 | PGR | 1.271733 |
520 | 3115 | MDM2 | 1.274517 |
521 | 3126 | KIF3B | 1.274522 |
522 | 3095 | KIF2C | 1.274859 |
523 | 2947 | 1.277515 | |
524 | 3408 | PIN1 | 1.278984 |
525 | 3657 | PCTK1 | 1.279578 |
526 | 3211 | BUB1B | 1.282741 |
527 | 3643 | DDR2 | 1.28316 |
528 | 3449 | TBK1 | 1.285291 |
529 | 3669 | NTRK3 | 1.285519 |
530 | 3200 | REL | 1.285524 |
531 | 3729 | RYK | 1.291213 |
532 | 3691 | 1.291243 | |
533 | 3214 | CENPF | 1.291507 |
534 | 3801 | PSEN1 | 1.291634 |
535 | 2978 | 1.293122 | |
536 | 3141 | 1.294102 | |
537 | 3792 | ARHGEF1 | 1.294579 |
538 | 3477 | FGFR4 | 1.29633 |
539 | 3169 | NME1 | 1.298008 |
540 | 3693 | NEK9 | 1.299989 |
541 | 3583 | JUNB | 1.300395 |
542 | 3768 | ARAF1 | 1.302137 |
543 | 2975 | NEK4 | 1.302558 |
544 | 3221 | top3B | 1.30285 |
545 | 3478 | EPHA2 | 1.30329 |
546 | 3666 | PAK6 | 1.303653 |
547 | 2963 | MAP3K11 | 1.306508 |
548 | 3199 | NF2 | 1.307337 |
549 | 3724 | EPHA7 | 1.308035 |
550 | 3457 | BAD | 1.308937 |
551 | 3185 | VCAM1 | 1.309542 |
552 | 3244 | PRKCZ | 1.310965 |
553 | 3587 | VAV3 | 1.31294 |
554 | 3712 | RPS6KA6 | 1.313627 |
555 | 3216 | CDC20 | 1.315701 |
556 | 3551 | STAT3 | 1.316414 |
557 | 3590 | ARHGEF2 | 1.316547 |
558 | 3659 | 1.317441 | |
559 | 3831 | CLSPN | 1.318145 |
560 | 3109 | KIF1C | 1.318847 |
561 | 3455 | MAP2K4 | 1.319428 |
562 | 3118 | 1.319892 | |
563 | 3709 | 1.320996 | |
564 | 3122 | ATSV | 1.321439 |
565 | 3809 | CDCA3 | 1.329173 |
566 | 3237 | CDC7 | 1.330145 |
567 | 3650 | 1.335065 | |
568 | 3382 | TUBB | 1.336093 |
569 | 3190 | WNT4 | 1.336703 |
570 | 3591 | RALB | 1.338466 |
571 | 3091 | KIFC3 | 1.339318 |
572 | 3761 | WT1 | 1.340453 |
573 | 3832 | ATM | 1.343275 |
574 | 3154 | MADH4 | 1.343448 |
575 | 3002 | CRKL | 1.345404 |
576 | 2946 | 1.346714 | |
577 | 3389 | 1.347114 | |
578 | 3645 | CASK | 1.34749 |
579 | 3315 | CCT4 | 1.348613 |
580 | 3150 | BRCA2 | 1.34964 |
581 | 3474 | CSNK2A1 | 1.350654 |
582 | 3458 | ARAF1 | 1.351534 |
583 | 3528 | TCF3 | 1.354214 |
584 | 3529 | DTX1 | 1.357117 |
585 | 3559 | RAPIGDS1 | 1.359432 |
586 | 3721 | ANKRD3 | 1.361311 |
587 | 3819 | ?TACC3 | 1.367136 |
588 | 3578 | ?RREB1 | 1.367845 |
589 | 3245 | ?AXL | 1.367989 |
590 | 3543 | ?PDK2 | 1.368231 |
591 | 3352 | ?NR3C2 | 1.368397 |
592 | 3493 | ?MAPK9 | 1.368899 |
593 | 2958 | ?PRKACA | 1.371294 |
594 | 3435 | ?FLT3 | 1.371521 |
595 | 3316 | ?CCNA1 | 1.37217 |
596 | 3263 | ?CDC2 | 1.373177 |
597 | 3224 | ?FBXO5 | 1.374389 |
598 | 2701 | 1.378002 | |
599 | 3497 | ?EPHA8 | 1.378119 |
600 | 3255 | ?CDK7 | 1.379045 |
601 | 3766 | ?S100A2 | 1.379101 |
602 | 3690 | ?PRKAA2 | 1.383537 |
603 | 3152 | ?APC | 1.384859 |
604 | 3201 | ?RARA | 1.387549 |
605 | 3396 | ?HDAC10 | 1.391302 |
606 | 3363 | ?GART | 1.392568 |
607 | 2957 | ?TYK2 | 1.392639 |
608 | 3323 | ?CDK5R1 | 1.394769 |
609 | 3380 | ?TUBG2 | 1.401159 |
610 | 3233 | ?ROCK1 | 1.404806 |
611 | 3806 | ?C10orf3 | 1.405976 |
612 | 3614 | ?CTNND2 | 1.409777 |
613 | 3093 | ?PIK3CG | 1.41077 |
614 | 3763 | 1.412316 | |
615 | 3487 | ?MAP2K3 | 1.412348 |
616 | 3732 | ?CSNK2A2 | 1.414035 |
617 | 3110 | ?KIF13A | 1.414042 |
618 | 3789 | ?ELK1 | 1.414448 |
619 | 3786 | ?FRAP1 | 1.416676 |
620 | 3554 | ?PIK3R3 | 1.418107 |
621 | 3167 | ?S100A2 | 1.418532 |
622 | 3084 | ?KIF14 | 1.41956 |
623 | 3661 | ?FGR | 1.423887 |
624 | 3617 | ?CTNNBIP1 | 1.425161 |
625 | 2974 | ?NEK11 | 1.426945 |
626 | 3330 | ?CDK9 | 1.428872 |
627 | 3227 | ?NUMA1 | 1.432118 |
628 | 3734 | ?BMPR1B | 1.437299 |
629 | 3138 | ?KIF17 | 1.441566 |
630 | 3186 | ?ETS1 | 1.442612 |
631 | 3673 | ?DDR1 | 1.444582 |
632 | 3450 | ?MAP3K2 | 1.446117 |
633 | 3133 | 1.450561 | |
634 | 3598 | ?PCNA | 1.451899 |
635 | 3106 | ?KIF9 | 1.45407 |
636 | 3608 | ?MAP3K7IP1 | 1.455728 |
637 | 3376 | ?AGA | 1.457466 |
638 | 3443 | ?EPS8 | 1.460769 |
639 | 3102 | CKS2 | 1.464872 |
640 | 3409 | TTK | 1.465445 |
641 | 3346 | CCNK | 1.465948 |
642 | 3604 | TK2 | 1.466617 |
643 | 2921 | RPS6KA5 | 1.467418 |
644 | 3597 | SHMT2 | 1.468236 |
645 | 3499 | GRB2 | 1.469395 |
646 | 3406 | TERT | 1.482496 |
647 | 3158 | BRCA1 | 1.485158 |
648 | 3114 | PIK3CD | 1.485825 |
649 | 3575 | LATS2 | 1.487058 |
650 | 3390 | 1.487135 | |
651 | 3596 | SHMT1 | 1.487573 |
652 | 3514 | HRAS | 1.488051 |
653 | 3730 | TESK1 | 1.489848 |
654 | 3620 | AXIN2 | 1.491167 |
655 | 3619 | FRAT1 | 1.491691 |
656 | 3644 | EPHB1 | 1.492026 |
657 | 3117 | ATM | 1.498897 |
658 | 3541 | PKD2 | 1.5005 |
659 | 3607 | TLE1 | 1.501123 |
660 | 3229 | PRKCL1 | 1.502059 |
661 | 3104 | CDK4 | 1.502301 |
662 | 3684 | STK38L | 1.5024 |
663 | 3626 | DVL3 | 1.504253 |
664 | 2986 | ACVR2 | 1.510627 |
665 | 2971 | DAPK2 | 1.516585 |
666 | 3759 | 1.517994 | |
667 | 3636 | STAT2 | 1.519082 |
668 | 3611 | CTNNAL1 | 1.523973 |
669 | 3794 | WASL | 1.529001 |
670 | 2944 | MARK1 | 1.53037 |
671 | 3713 | PRKG2 | 1.535337 |
672 | 3087 | PTEN | 1.540121 |
673 | 3506 | 1.54045 | |
674 | 3191 | WNT2 | 1.553178 |
675 | 3202 | MCC | 1.554866 |
676 | 3210 | 1.555868 | |
677 | 3738 | PRKWNK3 | 1.559877 |
678 | 3096 | AKT1 | 1.560781 |
679 | 3308 | CDKN2B | 1.566367 |
680 | 3606 | CREBBP | 1.570055 |
681 | 3641 | TYRO3 | 1.574144 |
682 | 3758 | RAD51L1 | 1.575115 |
683 | 3192 | WNT1 | 1.579448 |
684 | 3705 | 1.591723 | |
685 | 3565 | RAB2 | 1.597556 |
686 | 3770 | TGFBR2 | 1.602887 |
687 | 3771 | PIK3CA | 1.605624 |
688 | 3314 | CCNG1 | 1.606354 |
689 | 3579 | PDZGEF2 | 1.609017 |
690 | 3603 | POLS | 1.609696 |
691 | 3589 | SOS1 | 1.610658 |
692 | 2938 | ALS2CR7 | 1.613751 |
693 | 3621 | CTNNA2 | 1.62379 |
694 | 3265 | RAF1 | 1.625904 |
695 | 3698 | ADRBK2 | 1.626836 |
696 | 3622 | FZD9 | 1.630823 |
697 | 3111 | KIF5B | 1.633474 |
698 | 3688 | GUCY2D | 1.63373 |
699 | 3489 | SRC | 1.633931 |
700 | 3320 | CCND3 | 1.636023 |
701 | 2970 | AATK | 1.636349 |
702 | 3562 | RASD1 | 1.636677 |
703 | 3728 | TIE | 1.637314 |
704 | 3827 | 1.638302 | |
705 | 3778 | VHL | 1.639913 |
706 | 3448 | 1.6515 | |
707 | 3426 | TK1 | 1.654609 |
708 | 2701 | 1.655539 | |
709 | 3331 | CDC25B | 1.661891 |
710 | 3371 | 1.664564 | |
711 | 2923 | ERN1 | 1.665407 |
712 | 3550 | PLCG1 | 1.667241 |
713 | 3803 | MPHOSPH1 | 1.668632 |
714 | 3333 | CCNT2 | 1.669848 |
715 | 3520 | NRAS | 1.670763 |
716 | 3121 | PIK3C2A | 1.675061 |
717 | 3264 | CDK3 | 1.681459 |
718 | 3785 | GRB2 | 1.681539 |
719 | 3205 | 1.682938 | |
720 | 3811 | 1.685119 | |
721 | 3507 | 1.688535 | |
722 | 2955 | PAK1 | 1.688691 |
723 | 3440 | FGFR3 | 1.695183 |
724 | 2994 | MATK | 1.696094 |
72S | 2967 | 1.703715 | |
726 | 3325 | CDKN1C | 1.703926 |
727 | 3545 | IRS2 | 1.705996 |
728 | 3492 | PRKCQ | 1.706638 |
729 | 3547 | FOXO1A | 1.710389 |
730 | 3530 | NOTCH1 | 1.711344 |
731 | 3810 | 1.723959 | |
732 | 3321 | CDKN3 | 1.724044 |
733 | 3453 | MAP2K2 | 1.737418 |
734 | 3793 | MAPRE1 | 1.738248 |
735 | 2941 | DYRK3 | 1.741034 |
736 | 3217 | 1.745349 | |
737 | 3451 | MAP3K4 | 1.753145 |
738 | 3442 | ERBB4 | 1.760041 |
739 | 3696 | 1.760172 | |
740 | 3701 | STK10 | 1.767448 |
741 | 3817 | 1.768117 | |
742 | 2912 | MPHOSPH1 | 1.771582 |
743 | 3001 | PRKY | 1.772697 |
744 | 3128 | AKT2 | 1.773864 |
745 | 2981 | ALK | 1.781796 |
746 | 3337 | CDKN1A | 1.781903 |
747 | 3802 | NOTCH3 | 1.787122 |
748 | 3735 | PRKACB | 1.790032 |
749 | 3183 | 1.793085 | |
750 | 3430 | STMN1 | 1.798292 |
751 | 3531 | 1.800094 | |
752 | 3515 | PDGFRB | 1.80459 |
753 | 3324 | CUL5 | 1.820969 |
754 | 3511 | 1.832738 | |
755 | 3472 | MAP3K5 | 1.834487 |
756 | 3428 | ECT2 | 1.84097 |
757 | 3642 | EPHB3 | 1.84828 |
758 | 3208 | ZW10 | 1.858453 |
759 | 3820 | 1.861643 | |
760 | 3225 | 1.868827 | |
761 | 3834 | CHEK1 | 1.869085 |
762 | 3510 | CDK4 | 1.869212 |
763 | 3795 | NR4A2 | 1.870845 |
764 | 3247 | 1.875796 | |
765 | 3521 | MET | 1.887521 |
766 | 3538 | NFKB2 | 1.892227 |
767 | 3818 | 1.900799 | |
768 | 3719 | BMPR2 | 1.919267 |
769 | 3144 | KIF4B | 1.924148 |
770 | 3355 | GUK1 | 1.925235 |
771 | 2956 | PRKCL2 | 1.929173 |
772 | 3198 | ICAM1 | 1.937953 |
773 | 3361 | IMPDH2 | 1.938577 |
774 | 3672 | SYK | 1.945812 |
775 | 3697 | CAMK2G | 1.946161 |
776 | 3415 | HSPCA | 1.94686 |
777 | 3505 | STK6 | 1.949702 |
778 | 3368 | top3A | 1.959095 |
779 | 3681 | SRPK1 | 1.963919 |
780 | 3613 | DVL1 | 1.984151 |
781 | 3720 | 1.996699 | |
782 | 2995 | PTK2 | 2.015836 |
783 | 3522 | KRAS2 | 2.026984 |
784 | 3436 | BRAF | 2.036457 |
785 | 3787 | FZD4 | 2.049799 |
786 | 3584 | RASAL2 | 2.089191 |
787 | 3098 | CENPE | 2.090255 |
788 | 3267 | CCNH | 2.096356 |
789 | 2931 | MAP4K3 | 2.11675 |
790 | 2962 | MAP4K2 | 2.12521 |
791 | 3790 | ERBB3 | 2.13688 |
792 | 3742 | RHOK | 2.142917 |
793 | 2948 | MYO3A | 2.173575 |
794 | 3629 | AXIN1 | 2.184253 |
795 | 3546 | INPP5D | 2.197591 |
796 | 3723 | 2.212338 | |
797 | 2973 | NEK1 | 2.222767 |
798 | 3512 | TGFBR1 | 2.223853 |
799 | 3135 | 2.223901 | |
800 | 3637 | STAT4 | 2.227212 |
801 | 3004 | MAP3K1 | 2.235803 |
802 | 3304 | CCNE2 | 2.239326 |
803 | 3129 | STK6 | 2.248154 |
804 | 3402 | HDAC4 | 2.253527 |
805 | 3627 | CTNNA1 | 2.28197 |
806 | 3537 | EIF4EBP1 | 2.322458 |
807 | 3704 | ACVR2B | 2.322634 |
808 | 3329 | CDC42 | 2.333632 |
809 | 3259 | MAPK8 | 2.334959 |
810 | 3689 | BLK | 2.340679 |
811 | 3241 | WEE1 | 2.35419 |
812 | 3137 | KIF26A | 2.359341 |
813 | 3612 | TCF1 | 2.413867 |
814 | 3532 | 2.468626 | |
815 | 3764 | NOTCH4 | 2.482525 |
816 | 3417 | HDAC3 | 2.485246 |
817 | 3120 | PIK3CB | 2.528659 |
818 | 3313 | CCNG2 | 2.568855 |
819 | 3722 | TLK2 | 2.571781 |
820 | 3136 | 2.916125 | |
821 | 3780 | MCM3 | 2.988111 |
822 | 3580 | ELK1 | 3.0307 |
823 | 3718 | PTK6 | 3.090027 |
824 | 3777 | ABL1 | 3.099871 |
825 | 3605 | FZD4 | 3.155698 |
826 | 3134 | 3.263194 | |
827 | 2929 | CHUK | 3.298485 |
828 | 3781 | SRC | 3.433423 |
829 | 3223 | 3.587036 | |
830 | 3706 | C20orf97 | 4.288466 |
Table III A is used for the siRNA sequence of the screening of DNA disrupting agent: the cis-platinum screening
The gene title | Serial ID | Adopted sequence is arranged | SEQ?ID?NO |
CHUK | NM_001278 | AAAGGCUGCUCACAAGUUCTT | 50 |
CHUK | NM_001278 | AGCUGCUCAACAAACCAGATT | 51 |
CHUK | NM_001278 | AUGAGGAACAGGGCAAUAGTT | 52 |
PRKACA | NM_002730 | GAAUGGGGUCAACGAUAUCTT | 53 |
PRKACA | NM_002730 | GGACGAGACUUCCUCUUGATT | 54 |
PRKACA | NM_002730 | GUGUGGCAAGGAGUUUUCUTT | 55 |
MAP4K2 | NM_004579 | GAAUCCUAAGAAGAGGCCGTT | 56 |
MAP4K2 | NM_004579 | GAGGAGGUCUUUCAUUGGGTT | 57 |
MAP4K2 | NM_004579 | GAUAGUCAAGCUAGACCCATT | 58 |
STK17B | NM_004226 | AUCCUCCUGUAAUGGAACCTT | 59 |
STK17B | NM_004226 | GAAGAGGACAGGAUUGUCGTT | 60 | |
STK17B | NM_004226 | GACCAACAGCAGAGAUAUGTT | 61 | |
ALK | NM_004304 | ACCAGAGACCAAAUGUCACTT | 62 | |
ALK | NM_004304 | AUAAGCCCACCAGCUUGUGTT | 63 | |
ALK | NM_004304 | UCAACACCGCUUUGCCGAUTT | 64 | |
FRK | NM_002031 | ACUAUAGACUUCCGCAACCTT | 65 | |
FRK | NM_002031 | CAGUAGAUUGCUGUGGCCUTT | 66 | |
FRK | NM_002031 | CUCCAUACAGCUUCUGAAGTT | 67 | |
MAP3K1 | AF042838 | UCACUUAGCAGCUGAGUCUTT | 68 | |
MAP3K1 | AF042838 | UUGACAGCACUGGUCAGAGTT | 69 | |
MAP3K1 | AF042838 | UUGGCAAGAACUUCUUGGCTT | 70 | |
KIF2C | NM_006845 | ACAAAAACGGAGAUCCGUCTT | 71 | |
KIF2C | NM_006845 | AUAAGCAGCAAGAAACGGCTT | 72 | |
KIF2C | NM_006845 | GAAUUUCGGGCUACUUUGGTT | 73 | |
CENPE | NM_001813 | GAAAAUGAAGCUUUGCGGGTT | 74 | |
CENPE | NM_001813 | GAAGAGAUCCCAGUGCUUCTT | 75 | |
CENPE | NM_001813 | UCUGAAAGUGACCAGCUCATT | 76 | |
STK6 | NM_003600 | ACAGUCUUAGGAAUCGUGCTT | 3 | |
STK6 | NM_003600 | GCACAAAAGCUUGUCUCCATT | 1 | |
STK6 | NM_003600 | UUGCAGAUUUUGGGUGGUCTT | 2 | |
KIF4B | AF241316 | CCUGCAGCAACUGAUUACCTT | 77 | |
KIF4B | AF241316 | GAACUUGAGAAGAUGCGAGTT | 78 | |
KIF4B | AF241316 | GAAGAGGCCCACUGAAGUUTT | 79 | |
BRCA2 | NM_000059 | CAAAUGGGCAGGACUCUUATT | 80 | |
BRCA2 | NM_000059 | CUGUUCAGCCCAGUUUGAATT | 81 | |
BRCA2 | NM_000059 | UCUCCAAGGAAGUUGUACCTT | 82 | |
APC | | ACCAAGUAUCCGCAAAAGGTT | 83 | |
APC | NM_000038 | AGACCUGUAUUAGUACGCCTT | 84 | |
APC | NM_000038 | CAAGCUUUACCCAGCCUGUTT | 85 | |
ATR | NM_001184 | GAAACUGCAGCUAUCUUCCTT | 86 | |
ATR | NM_001184 | GUUACAAUGAGGCUGAUGCTT | 87 | |
ATR | NM_001184 | UCACGACUCGCUGAACUGUTT | 88 | |
BRCA1 | NM_007296 | ACUUAGGUGAAGCAGCAUCTT | 89 | |
BRCA1 | NM_007296 | GGGCAGUGAAGACUUGAUUTT | 90 | |
BRCA1 | NM_007296 | UGAAGUGGGCUCCAGUAUUTT | 91 | |
DCC | NM_005215 | ACAUCGUGGUGCGAGGUUATT | 92 | |
DCC | NM_005215 | AUGAGCCGCCAAUUGGACATT | 93 | |
DCC | NM_005215 | AUGGCAAGUUUGGAAGGACTT | 94 | |
WNT1 | NM_005430 | ACGGCGUUUAUCUUCGCUATT | 95 | |
WNT1 | NM_005430 | CCCUCUUGCCAUCCUGAUGTT | 96 | |
WNT1 | NM_005430 | CUAUUUAUUGUGCUGGGUCTT | 97 | |
CHEK1 | NM_001274 | AUCGAUUCUGCUCCUCUAGTT | 98 | |
CHEK1 | NM_001274 | CUGAAGAAGCAGUCGCAGUTT | 99 | |
CHEK1 | NM_001274 | UGCCUGAAAGAGACUUGUGTT | 100 | |
WEE1 | NM_003390 | AUCGGCUCUGGAGAAUUUGTT | 101 | |
WEE1 | NM_003390 | CAAGGAUCUCCAGUCCACATT | 102 | |
WEE1 | NM_003390 | UGUACCUGUGUGUCCAUCUTT | 103 | |
NM_018492 | AGGACACUUUGGGUACCAGTT | 104 | ||
NM_018492 | GACCCUAAAGAUCGUCCUUTT | 105 | ||
NM_018492 | GCUGAGGAGAAUAUGCCUCTT | 106 | ||
MAPK8 | NM_139049 | CACCCGUACAUCAAUGUCUTT | 107 | |
MAPK8 | NM_139049 | GGAAUAGUAUGCGCAGCUUTT | 108 |
MAPK8 | NM_139049 | GUGAUUCAGAUGGAGCUAGTT | 109 |
CUL1 | NM_003592 | GACCGCAAACUACUGAUUCTT | 110 |
CUL1 | NM_003592 | GCCAGCAUGAUCUCCAAGUTT | 111 |
CUL1 | NM_003592 | UAGACAUUGGGUUCGCCGUTT | 112 |
CCNG2 | NM_004354 | CCUCGAGAAAAAGGGCUGATT | 113 |
CCNG2 | NM_004354 | GCUCAGCUGAAAGCUUGCATT | 114 |
CCNG2 | NM_004354 | UGCCUAGCCGAGUAUUCUUTT | 115 |
CDC42 | NM_044472 | ACCUUAUGGAAAAGGGGUGTT | 116 |
CDC42 | NM_044472 | CCAUCCUGUUUGAAAGCCUTT | 117 |
CDC42 | NM_044472 | CCCAAAAGGAAGUGCUGUATT | 118 |
CDC25B | NM_021874 | AGGAUGAUGAUGCAGUUCCTT | 119 |
CDC25B | NM_021874 | GACAAGGAGAAUGUGCGCUTT | 120 |
CDC25B | NM_021874 | GAGCCCAGUCUGUUGAGUUTT | 121 |
top2B | NM_001068 | ACAUUCCCUGGAGUGUACATT | 122 |
top2B | NM_001068 | GAGGAUUUAGCGGCAUUUGTT | 123 |
top2B | NM_001068 | GCUGCUGGACUGCAUAAAGTT | 124 |
IMPDH2 | NM_000884 | AGAGGGAAGACUUGGUGGUTT | 125 |
IMPDH2 | NM_000884 | CACUCAUGCCAGGACAUUGTT | 126 |
IMPDH2 | NM_000884 | GAAGAAUCGGGACUACCCATT | 127 |
NM_007027 | ACUCACAGAAAAACCGUCGTT | 128 | |
NM_007027 | AUGAUGGGCGGACGAGUAUTT | 129 | |
NM_007027 | GAGUCAGCACCAUCAAAUGTT | 130 | |
HDAC4 | NM_006037 | AGAGGACGUUUUCUACGGCTT | 131 |
HDAC4 | NM_006037 | AUCUGUUUGCAAGGGGAAGTT | 132 |
HDAC4 | NM_006037 | CAAGAUCAUCCCCAAGCCATT | 133 |
TERT | NM_003219 | CACCAAGAAGUUCAUCUCCTT | 134 |
TERT | NM_003219 | GAGUGUCUGGAGCAAGUUGTT | 135 |
TERT | NM_003219 | GUUUGGAAGAACCCCACAUTT | 136 |
BRAF | NM_004333 | ACACUUGGUAGACGGGACUTT | 137 |
BRAF | NM_004333 | GUCAAUCAUCCACAGAGACTT | 138 |
BRAF | NM_004333 | UUGCAUGUGGAAGUGUUGGTT | 139 |
ERBB4 | NM_005235 | GAGUACUCUAUAGUGGCCUTT | 140 |
ERBB4 | NM_005235 | GCUUCCCAGUCCAAAUGACTT | 141 |
ERBB4 | NM_005235 | UGACAGUGGAGCAUGUGUUTT | 142 |
ABL2 | NM_007314 | AUCAGUGAUGUGGUGCAGATT | 143 |
ABL2 | NM_007314 | GACUCGGACACUGAAGAAATT | 144 |
ABL2 | NM_007314 | UGGCACAGCAGGUACUAAATT | 145 |
KRAS2 | NM_033360 | GAAAAGACUCCUGGCUGUGTT | 146 |
KRAS2 | NM_033360 | GGACUCUGAAGAUGUACCUTT | 147 |
KRAS2 | NM_033360 | GGCAUACUAGUACAAGUGGTT | 148 |
NM_021170 | AUCCUGGAGAUGACCGUGATT | 149 | |
NM_021170 | GCCGGUCAUGGAGAAGCGGTT | 150 | |
NM_021170 | UGGCCCUGAGACUGCAUCGTT | 151 | |
ELK1 | NM_005229 | GCCAUUCCUUUGUCUGCCATT | 152 |
ELK1 | NM_005229 | GUGAAAGUAGAAGGGCCCATT | 153 |
ELK1 | NM_005229 | UUCAAGCUGGUGGAUGCAGTT | 154 |
RASAL2 | NM_004841 | AGUACCAGGAUUCUUCAGCTT | 155 |
RASAL2 | NM_004841 | CUUAGUUCUGGGCCAUGUATT | 156 |
RASAL2 | NM_004841 | GACGCCACUGACAGUGAUUTT | 157 |
ARHGEF2 | NM_004723 | AGCUACACCACAGAUGCCATT | 158 |
ARHGEF2 | NM_004723 | GGACUUUGCAGCUGACUCUTT | 159 |
ARHGEF2 | NM_004723 | UAAAGGUUGGGGUGGCCAUTT | 160 |
FRAT1 | NM_005479 | AAGCUAAUGACGAGGAACCTT | 161 |
FRAT1 | NM_005479 | CCAUGGUGAAGUGCUUGGATT | 162 |
FRAT1 | NM_005479 | UAACAGCUGCAAUUCCCUGTT | 163 |
CTNNA2 | NM_004389 | CCUGAUGAAUGCUGUUGUCTT | 164 |
CTNNA2 | NM_004389 | GCACAAUACGGUGACCAAUTT | 165 |
CTNNA2 | NM_004389 | UCACAUCUUGGAGGAUGUGTT | 166 |
AXIN1 | AF009674 | GAAAGUGAGCGACGAGUUUTT | 167 |
AXIN1 | AF009674 | GUGCCUUCAACACAGCUUGTT | 168 |
AXIN1 | AF009674 | UGAAUAUCCAAGAGCAGGGTT | 169 |
EPHB3 | NM_004443 | GAAGAUCCUGAGCAGUAUCTT | 170 |
EPHB3 | NM_004443 | GCUGCAGCAGUACAUUGCUTT | 171 |
EPHB3 | NM_004443 | UACCCUGGACAAGCUCAUCTT | 172 |
DDR1 | NM_013994 | AACAAGAGGACACAAUGGCTT | 173 |
DDR1 | NM_013994 | AGAGGUGAAGAUCAUGUCGTT | 174 |
DDR1 | NM_013994 | UCGCAGACUUUGGCAUGAGTT | 175 |
CLK2 | NM_003993 | AUCGUUAGCACCUUAGGAGTT | 176 |
CLK2 | NM_003993 | CCCCUGCCUUGUACAUAAUTT | 177 |
CLK2 | NM_003993 | GUACAAGGAAGCAGCUCGATT | 178 |
C20orf97 | NM_021158 | AGUCCCAGGUGGGACUCUUTT | 179 |
C20orf97 | NM_021158 | CUGGCAUCCUUGAGCUGACTT | 180 |
C20orf97 | NM_021158 | GACUGUUCUGGAAUGAGGGTT | 181 |
X95425 | ACUGCCAGGAGUAAGAACUTT | 182 | |
X95425 | CUAUUACUGCAGAGGGCUUTT | 183 | |
X95425 | UGCAUCCUGCAGAGUAUCUTT | 184 | |
RPS6KA6 | NM_014496 | CCUCCUUUCAAACCUGCUUTT | 185 |
RPS6KA6 | NM_014496 | GAGGUUCUGUUUACAGAGGTT | 186 |
RPS6KA6 | NM_014496 | UCAGCCAGUGCAGAUUCAATT | 187 |
AB002301 | AGACAAAGAGGGGACCUUCTT | 188 | |
AB002301 | GAAAGUCUAUCCGAAGGCUTT | 189 | |
AB002301 | UGCCUCCCUGAAACUUCGATT | 190 | |
GPRK6 | NM_002082 | AAGCAAGAAAUGGCGGCAGTT | 191 |
GPRK6 | NM_002082 | GAGCUGAAUGUCUUUGGGCTT | 192 |
GPRK6 | NM_002082 | UGUAUAUAGCGACCAGAGCTT | 193 |
GSK3A | NM_019884 | CUUCAGUGCUGGUGAACUCTT | 194 |
GSK3A | NM_019884 | GCUGGACCACUGCAAUAUUTT | 195 |
GSK3A | NM_019884 | GUGGCUUACACGGACAUCATT | 196 |
RAD51L1 | NM_133510 | AACAGGACCGUACUGCUUGTT | 197 |
RAD51L1 | NM_133510 | GAAGCCUUUGUUCAGGUCUTT | 198 |
RAD51L1 | NM_133510 | GAGAGGCAUCCUCCUUGAATT | 199 |
NOTCH4 | NM_004557 | CCAGCACUGACUACUGUGUTT | 200 |
NOTCH4 | NM_004557 | GGAACUCGAUGCUUGUCAGTT | 201 |
NOTCH4 | NM_004557 | UGCGAGGAAGAUACGGAGUTT | 202 |
MCM3 | NM_002388 | GCAGAUGAGCAAGGAUGCUTT | 203 |
MCM3 | NM_002388 | GUACAUCCAUGUGGCCAAATT | 204 |
MCM3 | NM_002388 | UGGGUCAUGAAAGCUGCCATT | 205 |
FZD4 | NM_012193 | AGAACCUCGGCUACAACGUTT | 206 |
PZD4 | NM_012193 | UCCGCAUCUCCAUGUGCCATT | 207 |
FZD4 | NM_012193 | UCGGCUACAACGUGACCAATT | 208 |
Table III B is used for the siRNA sequence of the screening of DNA disrupting agent: the Zorubicin screening
Gene symbol | Serial ID | Adopted sequence is arranged | ?SEQ?ID?NO |
AATK | AB014541 | CGCAAGAAGAAGGCCGUGUTT | ?209 |
AATK | AB014541 | CGCUGGUGCAAUGUUUUCUTT | ?210 |
AATK | AB014541 | GAAUCCCUACCGAGACUCUTT | ?211 |
ABL1 | NM_007313 | AAACCUCUACACGUUCUGCTT | ?212 |
ABL1 | NM_007313 | CUAAAGGUGAAAAGCUCCGTT | ?213 |
ABL1 | NM_007313 | UCCUGGCAAGAAAGCUUGATT | ?214 |
ACVR2 | NM_001616 | AAGAUGGCCACAAACCUGCTT | ?215 |
ACVR2 | NM_001616 | AGAUAAACGGCGGCAUUGUTT | ?216 |
ACVR2 | NM_001616 | GACAUGCAGGAAGUUGUUGTT | ?217 |
ACVR2B | NM_001106 | CGGGAGAUCUUCAGCACACTT | ?218 |
ACVR2B | NM_001106 | GAGAUUGGCCAGCACCCUUTT | ?219 |
ACVR2B | NM_001106 | GCCCAGGACAUGAGUGUCUTT | ?220 |
ADRBK2 | NM_005160 | CGAGGAUGAGGCAUCUGAUTT | ?221 |
ADRBK2 | NM_005160 | CUGAAGUCCCUUUUGGAGGTT | ?222 |
ADRBK2 | NM_005160 | GAACUUCCCUUUGGUCAUCTT | ?223 |
AKT1 | NM_005163 | GCUGGAGAACCUCAUGCUGTT | ?224 |
AKT1 | NM_005163 | AGACGUUUUUGUGCUGUGGTT | ?225 |
AKT1 | NM_005163 | CGCACCUUCCAUGUGGAGATT | ?226 |
AKT2 | NM_001626 | AGAUGGCCACAUCAAGAUCTT | ?227 |
AKT2 | NM_001626 | GUCAUCAUUGCCAAGGAUGTT | ?228 |
AKT2 | NM_001626 | UGCCAGCUGAUGAAGACCGTT | ?229 |
ALK | NM_004304 | ACCAGAGACCAAAUGUCACTT | ?230 |
ALK | NM_004304 | AUAAGCCCACCAGCUUGUGTT | ?231 |
ALK | NM_004304 | UCAACACCGCUUUGCCGAUTT | ?232 |
ALS2CR7 | NM_139158 | CUGGCUGAUUUUGGUCUUGTT | ?233 |
ALS2CR7 | NM_139158 | GCCUUCAUGUUGUCUGGAATT | ?234 |
ALS2CR7 | NM_139158 | UCCACACCAAAGAGACACUTT | ?235 |
AXIN1 | AF009674 | GAAAGUGAGCGACGAGUUUTT | ?236 |
AXIN1 | AF009674 | GUGCCUUCAACACAGCUUGTT | ?237 |
AXIN1 | AF009674 | UGAAUAUCCAAGAGCAGGGTT | ?238 |
BLK | NM_001715 | AGUCACGAGCGUUCGAAAATT | ?239 |
BLK | NM_001715 | CAACAUGAAGGUGGCCAUUTT | ?240 |
BLK | NM_001715 | GCACUAUAAGAUCCGCUGCTT | ?241 |
BMPR2 | NM_001204 | CAAAUCUGUGAGCCCAACATT | ?242 |
BMPR2 | NM_001204 | CAAGAUGUUCUUGCACAGGTT | ?243 |
BMPR2 | NM_001204 | GAACGGCUAUGUGCGUUUATT | ?244 |
BRAF | NM_004333 | ACACUUGGUAGACGGGACUTT | ?245 |
BRAF | NM_004333 | GUCAAUCAUCCACAGAGACTT | ?246 |
BRAF | NM_004333 | UUGCAUGUGGAAGUGUUGGTT | ?247 |
C20orf97 | NM_021158 | AGUCCCAGGUGGGACUCUUTT | ?248 |
C20orf97 | NM_021158 | CUGGCAUCCUUGAGCUGACTT | ?249 |
C20orf97 | NM_021158 | GACUGUUCUGGAAUGAGGGTT | ?250 |
CAMK2G | BC021269 | GACAUUGUGGCCAGAGAGUTT | ?251 |
CAMK2G | BC021269 | GAUGAGGACCUCAAAGUGCTT | ?252 |
CAMK2G | BC021269 | GGCUGGAGCCUAUGAUUUCTT | ?253 |
CCND3 | NM_001760 | AAAGCAUGCCCAGACCUUUTT | ?254 |
CCND3 | NM_001760 | AAGGAUCUUUGUGGCCAAGTT | ?255 |
CCND3 | NM_001760 | CUACCUGGAUCGCUACCUGTT | ?256 |
CCNE2 | NM_057749 | CCACAGAUGAGGUCCAUACTT | ?257 |
CCNE2 | NM_057749 | CUGGGGCUUUCUUGACAUGTT | ?258 |
CCNE2 | NM_057749 | GUGGUUAAGAAAGCCUCAGTT | ?259 |
CCNG1 | NM_004060 | AUGGAUUGUUUCUGGGCGUTT | 260 |
CCNG1 | NM_004060 | CUAUCAGUCUUCCCACAGCTT | 261 |
CCNG1 | NM_004060 | CUUGCCACUUGAAAGGAGATT | 262 |
CCNG2 | NM_004354 | CCUCGAGAAAAAGGGCUGATT | 263 |
CCNG2 | NM_004354 | GCUCAGCUGAAAGCUUGCATT | 264 |
CCNG2 | NM_004354 | UGCCUAGCCGAGUAUUCUUTT | 265 |
CCNH | NM_001239 | GACCCGCUAUCCCAUAUUGTT | 266 |
CCNH | NM_001239 | GCCAGCAAUGCCAAGAUCUTT | 267 |
CCNH | NM_001239 | UUGCCCUGACUGCCAUUUUTT | 268 |
CCNT2 | NM_058241 | AGCGCCAGUAAAGAAGAACTT | 269 |
CCNT2 | NM_058241 | AGGGCAGCCAGUUGUCAUUTT | 270 |
CCNT2 | NM_058241 | CCACCACUCCAAAAUGAGCTT | 271 |
CDC25B | NM_021874 | AGGAUGAUGAUGCAGUUCCTT | 272 |
CDC25B | NM_021874 | GACAAGGAGAAUGUGCGCUTT | 273 |
CDC25B | NM_021874 | GAGCCCAGUCUGUUGAGUUTT | 274 |
CDC42 | NM_044472 | ACCUUAUGGAAAAGGGGUGTT | 275 |
CDC42 | NM_044472 | CCAUCCUGUUUGAAAGCCUTT | 276 |
CDC42 | NM_044472 | CCCAAAAGGAAGUGCUGUATT | 277 |
CDK3 | NM_001258 | CGAGAGGAAGCUCUAUCUGTT | 278 |
CDK3 | NM_001258 | GAGAGGAUGCAUCUGGGGATT | 279 |
CDK3 | NM_001258 | GAUCAGACUGGAUUUGGAGTT | 280 |
CDK4 | NM_000075 | CAGUCAAGCUGGCUGACUUTT | 281 |
CDK4 | NM_000075 | GCGAAUCUCUGCCUUUCGATT | 282 |
CDK4 | NM_000075 | GGAUCUGAUGCGCCAGUUUTT | 283 |
CDK4 | NM_000075 | CCCUGGUGUUUGAGCAUGUTT | 284 |
CDK4 | NM_000075 | CUGACCGGGAGAUCAAGGUTT | 285 |
CDK4 | NM_000075 | GAGUGUGAGAGUCCCCAAUTT | 286 |
CDKN1A | NM_078467 | AACUAGGCGGUUGAAUGAGTT | 287 |
CDKN1A | NM_078467 | CAUACUGGCCUGGACUGUUTT | 288 |
CDKN1A | NM_078467 | GAUGGUGGCAGUAGAGGCUTT | 289 |
CDKN1C | NM_000076 | AAAAACCGGGAUUCCGGCCTT | 290 |
CDKN1C | NM_000076 | GCGCAAGAGAUCAGCGCCUTT | 291 |
CDKN1C | NM_000076 | GUGGACAGCGACUCGGUGCTT | 292 |
CDKN2B | NM_004936 | ACACAGAGAAGCGGAUUUCTT | 293 |
CDKN2B | NM_004936 | CUCCAAGAGGUGGGUAAUUTT | 294 |
CDKN2B | NM_004936 | UGUCUGCUGAGGAGUUAUGTT | 295 |
CDKN3 | NM_005192 | CCUGCCUUAAAAAUUACCGTT | 296 |
CDKN3 | NM_005192 | GAACUAAAGAGCUGUGGUATT | 297 |
CDKN3 | NM_005192 | GAGGAUCCGGGGCAAUACATT | 298 |
CENPE | NM_001813 | GAAAAUGAAGCUUUGCGGGTT | 299 |
CENPE | NM_001813 | GAAGAGAUCCCAGUGCUUCTT | 300 |
CENPE | NM_001813 | UCUGAAAGUGACCAGCUCATT | 301 |
CHEK1 | NM_001274 | CCAGUUGAUGUUUGGUCCUTT | 302 |
CHEK1 | NM_001274 | UCUCAGACUUUGGCUUGGCTT | 303 |
CHEK1 | NM_001274 | UUCUAUGGUCACAGGAGAGTT | 304 |
CHUK | NM_001278 | AAAGGCUGCUCACAAGUUCTT | 305 |
CHUK | NM_001278 | AGCUGCUCAACAAACCAGATT | 306 |
CHUK | NM_001278 | AUGAGGAACAGGGCAAUAGTT | 307 |
CREBBP | NM_004380 | GACAUCCCGAGUCUAUAAGTT | 308 |
CREBBP | NM_004380 | GCACAAGGAGGUCUUCUUCTT | 309 |
CREBBP | NM_004380 | UGGAGGAGAAUUAGGCCUUTT | 310 |
CTNNA1 | NM_001903 | CGUUCCGAUCCUCUAUACUTT | 311 |
CTNNA1 | NM_001903 | UGACAUCAUUGUGCUGGCCTT | 312 |
CTNNA1 | NM_001903 | UGACCAAAGAUGACCUGUGTT | 313 |
CTNNA2 | NM_004389 | CCUGAUGAAUGCUGUUGUCTT | 314 |
CTNNA2 | NM_004389 | GCACAAUACGGUGACCAAUTT | 315 |
CTNNA2 | NM_004389 | UCACAUCUUGGAGGAUGUGTT | 316 |
CTNNAL1 | NM_003798 | AAGUGUUGUUGCUGGCAGATT | 317 |
CTNNAL1 | NM_003798 | ACUUGAGAAGCUUUUGGGGTT | 318 |
CTNNAL1 | NM_003798 | CUAGAGGUUUUUGCUGCAGTT | 319 |
CUL5 | NM_003478 | AAGAGUGAGCUGGUCAAUGTT | 320 |
CUL5 | NM_003478 | AUUUUGGAGUGCUUGGGCATT | 321 |
CUL5 | NM_003478 | UGGGUAAACAGGGCAGCAATT | 322 |
DAPK2 | NM_014326 | GAAUAUUUUUGGGACGCCGTT | 323 |
DAPK2 | NM_014326 | UCCAAGAGGCUCUCAGACATT | 324 |
DAPK2 | NM_014326 | UCUCAGAAGGUCCUCCUGATT | 325 |
DVL1 | NM_004421 | GGAGGAGAUCUUUGAUGACTT | 326 |
DVL1 | NM_004421 | GUACGCCAGCAGCUUGCUGTT | 327 |
DVL1 | NM_004421 | UCGGAUCACACGGCACCGATT | 328 |
DVL3 | NM_004423 | ACCCCAGUGAGUUCUUUGUTT | 329 |
DVL3 | NM_004423 | CCUGGACAAUGACACAGAGTT | 330 |
DVL3 | NM_004423 | GUUCAUUUAAGCCUCAGGGTT | 331 |
DYRK3 | NM_003582 | CCAUGUUUGCAUGGCCUUUTT | 332 |
DYRK3 | NM_003582 | CUUCUGGAGCAAUCCAAACTT | 333 |
DYRK3 | NM_003582 | UCUUUGGAUGCCCUCCACATT | 334 |
ECT2 | NM_018098 | ACUGGCUAAAGAUGCUGUGTT | 335 |
ECT2 | NM_018098 | GACCAUGGGAAAAUUGUGGTT | 336 |
ECT2 | NM_018098 | GCUUAGUACAGCGGGUUGATT | 337 |
EIF4EBP1 | NM_004095 | CCACCCCUUCCUUAGGUUGTT | 338 |
EIF4EBP1 | NM_004095 | CUCACCUGUGACCAAAACATT | 339 |
EIF4EBP1 | NM_004095 | UAGCCCAGAAGAUAAGCGGTT | 340 |
ELK1 | NM_005229 | GCCAUUCCUUUGUCUGCCATT | 341 |
ELK1 | NM_005229 | GUGAAAGUAGAAGGGCCCATT | 342 |
ELK1 | NM_005229 | UUCAAGCUGGUGGAUGCAGTT | 343 |
EPHB3 | NM_004443 | GAAGAUCCUGAGCAGUAUCTT | 344 |
EPHB3 | NM_004443 | GCUGCAGCAGUACAUUGCUTT | 345 |
EPHB3 | NM_004443 | UACCCUGGACAAGCUCAUCTT | 346 |
ERBB3 | NM_001982 | CUUUCUGAAUGGGGAGCCUTT | 347 |
ERBB3 | NM_001982 | UACACACACCAGAGUGAUGTT | 348 |
ERBB3 | NM_001982 | UGACAGUGGAGCCUGUGUATT | 349 |
ERBB4 | NM_005235 | GAGUACUCUAUAGUGGCCUTT | 350 |
ERBB4 | NM_005235 | GCUUCCCAGUCCAAAUGACTT | 351 |
ERBB4 | NM_005235 | UGACAGUGGAGCAUGUGUUTT | 352 |
ERN1 | NM_001433 | AAGCCUUACGGUCAUGAUGTT | 353 |
ERN1 | NM_001433 | GAAUAAUGAAGGCCUGACGTT | 354 |
ERN1 | NM_001433 | GAUGAUUGCGAUGGAUCCUTT | 355 |
FGFR3 | NM_000142 | AACAUCAUCAACCUGCUGGTT | 356 |
FGFR3 | NM_000142 | CACUUCCAGCAUUUAGCUGTT | 357 |
FGFR3 | NM_000142 | CACUUCUUACGCAAUGCUUTT | 358 |
FOXO1A | NM_002015 | CUAUGCGUACUGCAUAGCATT | 359 |
FOXO1A | NM_002015 | GACAACGACACAUAGCUGGTT | 360 |
FOXO1A | NM_002015 | UACAAGGAACCUCAGAGCCTT | 361 |
FZD4 | NM_012193 | CCAUCUGCUUGAGCUACUUTT | 362 |
FZD4 | NM_012193 | GUUGACUUACCUGACGGACTT | 363 |
FZD4 | NM_012193 | UUGGCAAAGGCUCCUUGUATT | 364 |
FZD4 | NM_012193 | AGAACCUCGGCUACAACGUTT | 365 |
FZD4 | NM_012193 | UCCGCAUCUCCAUGUGCCATT | 366 |
FZD4 | NM_012193 | UCGGCUACAACGUGACCAATT | 367 |
FZD9 | NM_003508 | GACUUUCCAGACCUGGCAGTT | 368 |
FZD9 | NM_003508 | GAUCGGGGUCUUCUCCAUCTT | 369 |
FZD9 | NM_003508 | GGACUUCGCGCUGGUCUGGTT | 370 |
GRB2 | NM_002086 | AUACGUCCAGGCCCUCUUUTT | 371 |
GRB2 | NM_002086 | CGGGCAGACCGGCAUGUUUTT | 372 |
GRB2 | NM_002086 | UGCAGCACUUCAAGGUGCUTT | 373 |
GUCY2D | NM_000180 | GAAAUUCCCAGGGGAUCAGTT | 374 |
GUCY2D | NM_000180 | GACAGACCGGCUGCUUACATT | 375 |
GUCY2D | NM_000180 | GUCACGGAACUGCAUAGUGTT | 376 |
GUK1 | NM_000858 | CGGCAAAGAUUACUACUUUTT | 377 |
GUK1 | NM_000858 | GGAGCCCGGCCUGUUUGAUTT | 378 |
GUK1 | NM_000858 | UCAAGAAAGCUCAAAGGACTT | 379 |
HDAC3 | NM_003883 | CCCAGAGAUUUUUGAGGGATT | 380 |
HDAC3 | NM_003883 | UGCCUUCAACGUAGGCGAUTT | 381 |
HDAC3 | NM_003883 | UGGUACCUAUUAGGGAUGGTT | 382 |
HDAC4 | NM_006037 | AGAGGACGUUUUCUACGGCTT | 383 |
HDAC4 | NM_006037 | AUCUGUUUGCAAGGGGAAGTT | 384 |
HDAC4 | NM_006037 | CAAGAUCAUCCCCAAGCCATT | 385 |
HSPCA | NM_005348 | ACACCUGGAGAUAAACCCUTT | 386 |
HSPCA | NM_005348 | CCUAUGGGUCGUGGAACAATT | 387 |
HSPCA | NM_005348 | UAACCUUGGUACUAUCGCCTT | 388 |
ICAM1 | NM_000201 | CAGCUAAAACCUUCCUCACTT | 389 |
ICAM1 | NM_000201 | AACACAAAGGCCCACACUUTT | 390 |
ICAM1 | NM_000201 | CAGAGUGGAAGACAUAUGCTT | 391 |
1MPDH2 | NM_000884 | AGAGGGAAGACUUGGUGGUTT | 392 |
IMPDH2 | NM_000884 | CACUCAUGCCAGGACAUUGTT | 393 |
IMPDH2 | NM_000884 | GAAGAAUCGGGACUACCCATT | 394 |
INPP5D | NM_005541 | AGCAUUAAGAAGCCCAGUGTT | 395 |
INPP5D | NM_005541 | GAACAAGCACUCAGAGCAGTT | 396 |
INPP5D | NM_005541 | UCCCAUCAACAUGGUGUCCTT | 397 |
IRS2 | NM_003749 | CACAGCCGUUCGAUGUCCATT | 398 |
IRS2 | NM_003749 | GUACAUCGCCAUCGACGUGTT | 399 |
IRS2 | NM_003749 | GUACCUGAUCGCCCUCUACTT | 400 |
KIF26A | XM_050278 | AUGCGGAAUUUGCCGUGGGTT | 401 |
KIF26A | XM_050278 | GCACAAGCACCUGUGUGAGTT | 402 |
KIF26A | XM_050278 | GUCGUACACCAUGAUCGGGTT | 403 |
KIF4B | AF241316 | CCUGCAGCAACUGAUUACCTT | 404 |
KIF4B | AF241316 | GAACUUGAGAAGAUGCGAGTT | 405 |
KIF4B | AF241316 | GAAGAGGCCCACUGAAGUUTT | 406 |
KIFB | NM_004521 | AAUGCAUCUCGUGAUCGCATT | 407 |
KIF5B | NM_004521 | AGACAGUUGGAGGAAUCUGTT | 408 |
KIF5B | NM_004521 | AUCGGCAACUUUAGCGAGUTT | 409 |
KRAS2 | NM_033360 | GAAAAGACUCCUGGCUGUGTT | 410 |
KRAS2 | NM_033360 | GGACUCUGAAGAUGUACCUTT | 411 |
KRAS2 | NM_033360 | GGCAUACUAGUACAAGUGGTT | 412 |
MAP2K2 | NM_030662 | ACCACACCUUCAUCAAGCGTT | 413 |
MAP2K2 | NM_030662 | AGUCAGCAUCGCGGUUCUCTT | 414 |
MAP2K2 | NM_030662 | GAAGGAGAGCCUCACAGCATT | 415 |
MAP3K1 | AF042838 | UCACUUAGCAGCUGAGUCUTT | 416 |
MAP3K1 | AF042838 | UUGACAGCACUGGUCAGAGTT | 417 |
MAP3K1 | AF042838 | UUGGCAAGAACUUCUUGGCTT | 418 |
MAP3K4 | NM_005922 | AGAACGAUCGUCCAGUGGATT | 419 |
MAP3K4 | NM_005922 | GGUACCUCGAUGCCAUAGUTT | 420 |
MAP3K4 | NM_005922 | UUUUGGACUAGUGCGGAUGTT | 421 |
MAP3K5 | NM_005923 | AGAAUUGGCAGCUGAGUUGTT | 422 |
MAP3K5 | NM_005923 | UGCAGCAGCUAUUGCACUUTT | 423 |
MAP3K5 | NM_005923 | UGUACAGCUUGGAAGGAUGTT | 424 |
MAP4K2 | NM_004579 | GAAUCCUAAGAAGAGGCCGTT | 425 |
MAP4K2 | NM_004579 | GAGGAGGUCUUUCAUUGGGTT | 426 |
MAP4K2 | NM_004579 | GAUAGUCAAGCUAGACCCATT | 427 |
MAP4K3 | NM_003618 | AAUGGGAUGCUGGCAAUGATT | 428 |
MAP4K3 | NM_003618 | AUCCUUACACGGGCCAUAATT | 429 |
MAP4K3 | NM_003618 | CUGGACCUCUGUCAGAACUTT | 430 |
MAPK8 | NM_139049 | CACCCGUACAUCAAUGUCUTT | 431 |
MAPK8 | NM_139049 | GGAAUAGUAUGCGCAGCUUTT | 432 |
MAPK8 | NM_139049 | GUGAUUCAGAUGGAGCUAGTT | 433 |
MAPRE1 | NM_012325 | GAGUAUUAACAGCCUGGACTT | 434 |
MAPRE1 | NM_012325 | GCUAAGCUAGAACACGAGUTT | 435 |
MAPRE1 | NM_012325 | UAGAGGAUGUGUUUCAGCCTT | 436 |
MARK1 | NM_018650 | ACAACAGCACUCUUCAGUCTT | 437 |
MARK1 | NM_018650 | CUGCGAGAGCGAGUUUUACTT | 438 |
MARK1 | NM_018650 | UGUGUAUUCUGGAGGUAGCTT | 439 |
MATK | NM_002378 | AGCGGAAACACGGGACCAATT | 440 |
MATK | NM_002378 | GUGUGAUGUGACAGCCCAGTT | 441 |
MATK | NM_002378 | UGUCACUGAAAGAGGUGUCTT | 442 |
MCC | NM_002387 | AGUUGAGGAGGUUUCUGCATT | 443 |
MCC | NM_002387 | GACUUAGAGCUGGGAAUCUTT | 444 |
MCC | NM_002387 | GGAUUAUAUCCAGCAGCUCTT | 445 |
MCM3 | NM_002388 | GCAGAUGAGCAAGGAUGCUTT | 446 |
MCM3 | NM_002388 | GUACAUCCAUGUGGCCAAATT | 447 |
MCM3 | NM_002388 | UGGGUCAUGAAAGCUGCCATT | 448 |
MET | NM_000245 | AUGCCUCUGGAGUGUAUUCTT | 449 |
MET | NM_000245 | AUGCGCCCAUCCUUUUCUGTT | 450 |
MET | NM_000245 | GAUCUGGGCAGUGAAUUAGTT | 451 |
MPHOSPH1 | NM_016195 | AGAGGAACUCUCUGCAAGCTT | 452 |
MPHOSPH1 | NM_016195 | CUGAAGAAGCUACUGCUUGTT | 453 |
MPHOSPH1 | NM_016195 | GACAUGCGAAUGACACUAGTT | 454 |
MPHOSPH1 | NM_016195 | AAGUUUGUGUCCCAGACACTT | 455 |
MPHOSPH1 | NM_016195 | AAUGGCAGUGAAACACCCUTT | 456 |
MPHOSPH1 | NM_016195 | AUGAAGGAGAGUGAUCACCTT | 457 |
MYO3A | NM_017433 | AAAGCUACCGAUGUCAGGGTT | 458 |
MYO3A | NM_017433 | AAAUCCCGAGUUAUCCACCTT | 459 |
MYO3A | NM_017433 | GGCUAAUGAAAGGUGCUGGTT | 460 |
NEK1 | AB067488 | AAGUGACAUUUGGGCUCUGTT | 461 |
NEK1 | AB067488 | AUGCACGUGCUGCUGUACUTT | 462 |
NEK1 | AB067488 | GAAGGACCUUCUGAUUCUGTT | 463 |
NFKB2 | NM_002502 | AGGAUUCUCAUGGGAAGGGTT | 464 |
NFKB2 | NM_002502 | GAAGAACAUGAUGGGGACUTT | 465 |
NFKB2 | NM_002502 | GAUUGAGCGGCCUGUAACATT | 466 |
NOTCH1 | AF308602 | AGGCAAGCCCUGCAAGAAUTT | 467 |
NOTCH1 | AF308602 | AUAUCGACGAUUGUCCAGGTT | 468 |
NOTCH1 | AF308602 | CACUUACACCUGUGUGUGCTT | 469 |
NOTCH3 | NM_000435 | AAUGGCUUCCGCUGCCUCUTT | 470 |
NOTCH3 | NM_000435 | GAACAUGGCCAAGGGUGAGTT | 471 |
NOTCH3 | NM_000435 | GAGUCUGGGACCUCCUUCUTT | 472 |
NOTCH4 | NM_004557 | CCAGCACUGACUACUGUGUTT | 473 |
NOTCH4 | NM_004557 | GGAACUCGAUGCUUGUCAGTT | 474 |
NOTCH4 | NM_004557 | UGCGAGGAAGAUACGGAGUTT | 475 |
NR4A2 | NM_006186 | AAGGCCGGAGAGGUCGUUUTT | 476 |
NR4A2 | NM_006186 | CAUCGACAUUUCUGCCUUCTT | 477 |
NR4A2 | NM_006186 | GUCACAUGGGCAGAGAUAGTT | 478 |
NRAS | NM_002524 | AAGAGCCACUUUCAAGCUGTT | 479 |
NRAS | NM_002524 | AGUAGCAACUGCUGGUGAUTT | 480 |
NRAS | NM_002524 | CCUCUACAGGGAGCAGAUUTT | 481 |
PAK1 | NM_002576 | CCGCUGUCUCGAUAUGGAUTT | 482 |
PAK1 | NM_002576 | GGACCGAUUUUACCGAUCCTT | 483 |
PAK1 | NM_002576 | UGGAUGGCUCUGUCAAGCUTT | 484 |
PDGFRB | NM_002609 | AAAGAAGUACCAGCAGGUGTT | 485 |
PDGFRB | NM_002609 | UCCAUCCACCAGAGUCUAGTT | 486 |
PDGFRB | NM_002609 | UUUGCUGAGCUGCAUCGGATT | 487 |
PDZGEF2 | NM_016340 | AACCCUCAUCCACAGGUGATT | 488 |
PDZGEF2 | NM_016340 | CCGACUGAGUACAUCGAUGTT | 489 |
PDZGEF2 | NM_016340 | GCCAGAUUCGACUGAUUGUTT | 490 |
PIK3C2A | NM_002645 | AACGAGGAAUCCGACAUUCTT | 491 |
PIK3C2A | NM_002645 | UGAUGAGCCCAUCCUUUCATT | 492 |
PIK3C2A | NM_002645 | UGCUUCAACGGAUGUAGCATT | 493 |
PIK3CA | NM_006218 | AGGUGCACUGCAGUUCAACTT | 494 |
PIK3CA | NM_006218 | UGGCUUUGAAUCUUUGGCCTT | 495 |
PIK3CA | NM_006218 | UUCAGCUAGUACAGGUCCUTT | 496 |
PIK3CB | NM_006219 | AAGUUCAUGUCAGGGCUGGTT | 497 |
PIK3CB | NM_006219 | AAUGCGCAAAUUCAGCGAGTT | 498 |
PIK3CB | NM_006219 | CAAAGAUGCCCUUCUGAACTT | 499 |
PKD2 | NM_000297 | CGGCUAGUACGUGAAGAGUTT | 500 |
PKD2 | NM_000297 | CUUAUAGUGGAGCUGGCUATT | 501 |
PKD2 | NM_000297 | GAUAGCGGACAUAGCUCCATT | 502 |
PLCG1 | NM_002660 | ACUACGUGGAAGAGAUGGUTT | 503 |
PLCG1 | NM_002660 | AGGCAAGAAGUUCCUUCAGTT | 504 |
PLCG1 | NM_002660 | GGAGAAUGGUGACCUCAGUTT | 505 |
POLS | NM_006999 | ACAGAGACGCCGAAAGUACTT | 506 |
POLS | NM_006999 | CUAGCGACAUAGACCUGGUTT | 507 |
POLS | NM_006999 | GCAAAUGAAUUGGCCUGGCTT | 508 |
PRKACB | NM_002731 | AAACCCUUGGAACAGGUUCTT | 509 |
PRKACB | NM_002731 | CAAGAUGACAUCUGAGCUCTT | 510 |
PRKACB | NM_002731 | UGUCUGAUCGAUCAUGCAGTT | 511 |
PRKCL1 | NM_002741 | CACAAGAUCGUCUACAGGGTT | 512 |
PRKCL1 | NM_002741 | CACCAGUGAAGUCAGCACUTT | 513 |
PRKCL1 | NM_002741 | GAUUUCAAGUUCCUGGCGGTT | 514 |
PRKCL2 | NM_006256 | AAGUGGCUCCUCAUUGUACTT | 515 |
PRKCL2 | NM_006256 | AUCAUUCUGGCACCUUCAGTT | 516 |
PRKCL2 | NM_006256 | GUAAAAGCGGAAGUAGUCGTT | 517 |
PRKCQ | NM_006257 | AAAUGAAGCAAGGCCGCCATT | 518 |
PRKCQ | NM_006257 | ACGGAGUACAGACGUCUCATT | 519 |
PRKCQ | NM_006257 | GGAAGCAAAGGACCUUCUGTT | 520 | |
PRKG2 | NM_006259 | AAGACUGGAUCCUCAGCAGTT | 521 | |
PRKG2 | NM_006259 | CAAGUGCAUCCAGCUGAACTT | 522 | |
PRKG2 | NM_006259 | GAAAUUCCUGCACAAUGGGTT | 523 | |
PRKWNK3 | AJ409088 | ACCAAGCAGCCAGCUAUACTT | 524 | |
PRKWNK3 | AJ409088 | CUAAUGACAUCUGGGACCUTT | 525 | |
PRKWNK3 | AJ409088 | CUACGAAGGAAAACGUCAGTT | 526 | |
PRKY | NM_002760 | AGACAGUGAAGCUGGUUGUTT | 527 | |
PRKY | NM_002760 | GAAUUUCUGAGGACGAGCUTT | 528 | |
PRKY | | UCAGAUUUGGGCCAGAGUUTT | 529 | |
PTEN | NM_000314 | UGGAGGGGAAUGCUCAGAATT | 530 | |
PTEN | NM_000314 | AAGGCAGCUAAAGGAAGUGTT | 531 | |
PTEN | NM_000314 | UAAAGAUGGCACUUUCCCGTT | 532 | |
PTK2 | NM_005607 | CUUGGACGAUGUAUUGGAGTT | 533 | |
PTK2 | NM_005607 | GACUGAAAAUGCUUGGGCATT | 534 | |
PTK2 | NM_005607 | UCCCACACAUCUUGCUGACTT | 535 | |
PTK6 | NM_005975 | AACACCCUCUGCAAAGUUGTT | 536 | |
PTK6 | NM_005975 | CCGUGGUUCUUUGGCUGCATT | 537 | |
PTK6 | NM_005975 | UCAGGCUUAUCCGAUGUGCTT | 538 | |
RAB2 | NM_002865 | AAUUGGCCCUCAGCAUGCUTT | 539 | |
RAB2 | NM_002865 | GAAGGUGAAGCUUUUGCACTT | 540 | |
RAB2 | NM_002865 | GAGGUUUCAGCCAGUGCAUTT | 541 | |
RAD51L1 | NM_133510 | AACAGGACCGUACUGCUUGTT | 542 | |
RAD51L1 | NM_133510 | GAAGCCUUUGUUCAGGUCUTT | 543 | |
RAD51L1 | NM_133510 | GAGAGGCAUCCUCCUUGAATT | 544 | |
RAF1 | NM_002880 | CACUCUCUACCGAAGAUCATT | 545 | |
RAF1 | NM_002880 | GAUCCUAAAGGUUGUCGACTT | 546 | |
RAF1 | NM_002880 | GGAAGCCAUUUGCAGUGCUTT | 547 | |
RASAL2 | NM_004841 | AGUACCAGGAUUCUUCAGCTT | 548 | |
RASAL2 | NM_004841 | CUUAGUUCUGGGCCAUGUATT | 549 | |
RASAL2 | NM_004841 | GACCCCACUGACAGUGAUUTT | 550 | |
RASD1 | NM_016084 | CAAAACCAAGGAGAACGUGTT | 551 | |
RASD1 | NM_016084 | CCUAAGGAGGACCUUUUUGTT | 552 | |
RASD1 | NM_016084 | GAAACCGUCAUGCCCGCUUTT | 553 | |
RHOK | NM_002929 | AGUACACAGCAGGUUCAUCTT | 554 | |
RHOK | NM_002929 | CGUGAAUGAGGAGAACCCUTT | 555 | |
RHOK | NM_002929 | GUUUAAGGAGGGGCCUGUGTT | 556 | |
SOS1 | NM_005633 | AUUGACCACCAGGUUUCUGTT | 557 | |
SOS1 | NM_005633 | CUUACAAAAGGGAGCACACTT | 558 | |
SOS1 | NM_005633 | UAUCAGACCGGACCUCUAUTT | 559 | |
SRC | NM_005417 | CAAUUCGUCGGAGGCAUCATT | 560 | |
SRC | NM_005417 | GCAGUGCCUGCCUAUGAAATT | 561 | |
SRC | NM_005417 | GGGGAGUUUGCUGGACUUUTT | 562 | |
SRC | NM_005417 | GAACCGGAUGCAGUUGAGCTT | 563 | |
SRC | NM_005417 | GCCGGAAUACAAGAACGGGTT | 564 | |
SRC | NM_005417 | GUGGCUCUUAUCCGCAUGATT | 565 | |
SRPK1 | NM_003137 | CGCUUAUGGAACGUGAUACTT | 566 | |
SRPK1 | NM_003137 | GCAACAGAAUGGCAGCGAUTT | 567 | |
SRPK1 | NM_003137 | GUUCUAAUCGGAUCUGGCUTT | 568 | |
STAT2 | NM_005419 | AAAGCCUGCAUCAGAGCUCTT | 569 | |
STAT2 | NM_005419 | AGUUAAUCUCCAGGAACGGTT | 570 | |
STAT2 | NM_005419 | UUUGCUCAGCCCAAACCUUTT | 571 |
STAT4 | NM_003151 | ACACAGAUCUGCCUCUAUGTT | 572 |
STAT4 | NM_003151 | CCCUACAAUAAAGGCCGGUTT | 573 |
STAT4 | NM_003151 | UUAGGAAGGUCCUUCAGGGTT | 574 |
STK10 | NM_005990 | AGACCAGUACUUCCUCCAGTT | 575 |
STK10 | NM_005990 | CCAUACUCAGAACUCCUCUTT | 576 |
STK10 | NM_005990 | GAAAAAGCAUCAGGGGGAATT | 577 |
STK38L | BC028603 | AGACACCUUGACAGAAGAGTT | 578 |
STK38L | BC028603 | CUCUGGGGAUUUCUCAAUGTT | 579 |
STK38L | BC028603 | GGAAGUAAAUGCAGGCCAGTT | 580 |
STK6 | NM_003600 | ACAGUCUUAGGAAUCGUGCTT | 581 |
STK6 | NM_003600 | GCACAAAAGCUUGUCUCCATT | 582 |
STK6 | NM_003600 | UUGCAGAUUUUGGGUGGUCTT | 583 |
STK6 | NM_003600 | CACCCAAAAGAGCAAGCAGTT | 584 |
STK6 | NM_003600 | CCUCCCUAUUCAGAAAGCUTT | 585 |
STK6 | NM_003600 | GACUUUGAAAUUGGUCGCCTT | 586 |
STMN1 | NM_005563 | AACUGCACAGUGCUGUUGGTT | 587 |
STMN1 | NM_005563 | UACCCAACGCACAAAUGACTT | 588 |
STMN1 | NM_005563 | UGGCUAGUACUGUAUUGGCTT | 589 |
SYK | NM_003177 | AGAACUGGGCUCUGGUAAUTT | 590 |
SYK | NM_003177 | AGAAGUUCGACACGCUCUGTT | 591 |
SYK | NM_003177 | GGAAAACCUCAUCAGGGAATT | 592 |
TCF1 | NM_000545 | AGCCGUGGUGGAGACCCUUTT | 593 |
TCF1 | NM_000545 | AGUCAAGGAGAAAUGCGGUTT | 594 |
TCF1 | NM_000545 | CCUCGUCACGGAGGUGCGUTT | 595 |
TGFBR1 | NM_004612 | GACAUGAUUCAGCCACAGATT | 596 |
TGFBR1 | NM_004612 | UUCCUCGAGAUAGGCCGUUTT | 597 |
TGFBR1 | NM_004612 | UUUGGGAGGUCAGUUGUUCTT | 598 |
TGFBR2 | NM_003242 | CCAGAAAUCCUGCAUGAGCTT | 599 |
TGFBR2 | NM_003242 | GCAGAACACUUCAGAGCAGTT | 600 |
TIE | NM_005424 | AAAAAGGGAUCUGGGGAUGTT | 601 |
TIE | NM_005424 | CGUGACGUUAAUGAACCUGTT | 602 |
TIE | NM_005424 | GAGCAACGGAUCCUACUUCTT | 603 |
TK1 | NM_003258 | AAGCACAGAGUUGAUGAGATT | 604 |
TK1 | NM_003258 | CCUUGCUGGGACUUGGAUCTT | 605 |
TK1 | NM_003258 | CGCCGGGAAGACCGUAAUUTT | 606 |
TLE1 | NM_005077 | AGAUGACAAGAAGCACCACTT | 607 |
TLE1 | NM_005077 | AGGAAGGUGGAUGAUAAGGTT | 608 |
TLE1 | NM_005077 | CACCUGUUUCCAAACCUUGTT | 609 |
TLK2 | NM_006852 | AGAGCUGGAUCAUCCCAGATT | 610 |
TLK2 | NM_006852 | AGGCGUUUAUUCGACGAUGTT | 611 |
TLK2 | NM_006852 | CACUUGACGGUUGUCCCUUTT | 612 |
top3A | NM_004618 | GAUCCUCCCUGUCUAUGAGTT | 613 |
top3A | NM_004618 | GCAAAGAAAUUGGACGAGGTT | 614 |
top3A | NM_004618 | GGCGAAAACAUCGGGUUUGTT | 615 |
TYRO3 | NM_006293 | GACUAACAAAGGCAGCUGUTT | 616 |
TYRO3 | NM_006293 | GCAGCUUGCAUGAAGGAGUTT | 617 |
TYRO3 | NM_006293 | UGCCCCUUUCCAACUGUCUTT | 618 |
VHL | NM_000551 | AGGAAAUAGGCAGGGUGUGTT | 619 |
VHL | NM_000551 | CAGAACCCAAAAGGGUAAGTT | 620 |
VHL | NM_000551 | GAUCUGGAAGACCACCCAATT | 621 |
WASL | NM_003941 | AAACAGGAGGUGUUGAAGCTT | 622 |
WASL | NM_003941 | AAGUGGAGCAGAACAGUCGTT | 623 |
WASL | NM_003941 | GGACAAUCCACAGAGAUCUTT | 624 |
WEE1 | NM_003390 | AUCGGCUCUGGAGAAUUUGTT | 625 |
WEE1 | NM_003390 | CAAGGAUCUCCAGUCCACATT | 626 |
WEE1 | NM_003390 | UGUACCUGUGUGUCCAUCUTT | 627 |
WNT1 | NM_005430 | ACGGCGUUUAUCUUCGCUATT | 628 |
WNT1 | NM_005430 | CCCUCUUGCCAUCCUGAUGTT | 629 |
WNT1 | NM_005430 | CUAUUUAUUGUGCUGGGUCTT | 630 |
WNT2 | NM_003391 | AUUUGCCCGCGCAUUUGUGTT | 631 |
WNT2 | NM_003391 | AACGGGCGAUUAUCUCUGGTT | 632 |
WNT2 | NM_003391 | AGAAGAUGAAUGGUCUGGCTT | 633 |
ZW10 | NM_004724 | ACAGUUGCAGGAGUUUUCCTT | 634 |
ZW10 | NM_004724 | CAAACUGUCAGGCAGCAUUTT | 635 |
ZW10 | NM_004724 | GCCAGCUUGCAAGAAAUUGTT | 636 |
XM_170783 | ACCGACACUUUGGCUUCCATT | 637 | |
XM_170783 | GAUGAGCGCGGGAAUGUUGTT | 638 | |
XM_170783 | UGGCCGAGGCCUUCAAGCUTT | 639 | |
XM_064050 | CAUCAAUCACUCUCUGCUGTT | 640 | |
XM_064050 | CUAACCCAGGAUGUUCAGGTT | 641 | |
XM_064050 | GACACUCACCAUGCUGAAATT | 642 | |
XM_066649 | AAGGGUGACUUUGUGUCCUTT | 643 | |
XM_066649 | ACCAGGAACAAACCUGUUGTT | 644 | |
XM_066649 | UUUGAAGGUGGCCCUCCUATT | 645 | |
NM_005200 | AUGAAGCCUCACCAGGACUTT | 646 | |
NM_005200 | CACUUUUCCCUCAACGAGGTT | 647 | |
NM_005200 | UAGUAGCAAAGCAGGAAGGTT | 648 | |
NM_139286 | GUUGUAGGAGGCAGUGAUGTT | 649 | |
NM_139286 | UCUCAAUUUGGAAGUCUUGTT | 650 | |
NM_139286 | UGAUCAAUGAUCGGAUUGGTT | 651 | |
NM_013366 | CGAUCUGCAGGCCAACAUCTT | 652 | |
NM_013366 | GAAGUAUGAGCAGCUCAAGTT | 653 | |
NM_013366 | GGACCUCUUCAUCAAUGAGTT | 654 | |
NM_014885 | ACCAGGAUUUGGAGUGGAUTT | 655 | |
NM_014885 | CAAGGCAUCCGUUAUAUCUTT | 656 | |
NM_014885 | GUGGCUGGAUUCAUGUUCCTT | 657 | |
NM_016263 | CCAGAUCCUUGUCUGGAAGTT | 658 | |
NM_016263 | CGACAACAAGCUGCUGGUCTT | 659 | |
NM_016263 | GAAGCUGUCCAUGUUGGAGTT | 660 | |
NM_013367 | AGCCAGCAGAUGUAAUUGGTT | 661 | |
NM_013367 | CAUUUCAAUGAGGCUCCAGTT | 662 | |
NM?013367 | GUCAUUUACAGAGUGGCUCTT | 663 | |
NM_018492 | AGGACACUUUGGGUACCAGTT | 664 | |
NM_018492 | GACCCUAAAGAUCGUCCUUTT | 665 | |
NM_018492 | GCUGAGGAGAAUAUGCCUCTT | 666 | |
NM_006087 | CGUGUACUACAACGAGGCCTT | 667 | |
NM_006087 | UCCCCUCUGACUCCAACUUTT | 668 | |
NM_006087 | CGAGGCACUCUACGACAUCTT | 669 | |
NM_016231 | GCAAUGAGGACAGCUUGUGTT | 670 | |
NM_016231 | UCUCCUUGUGAACAGCAACTT | 671 | |
NM_016231 | UGUAGCUUUCCACUGGAGUTT | 672 | |
XM_095827 | AAGGUCUUUACGCCAGUACTT | 673 | |
XM_095827 | GGAAUGUAUCCGAGCACUGTT | 674 | |
XM_095827 | UAAGCCUGGUGGUGAUCUUTT | 675 |
NM_145754 | CUCAAGGGAAAUAUCCGUGTT | 676 | ||
NM_145754 | GUGUGUUGUGCCUGCUGAATT | 677 | ||
NM_145754 | UCAGGCAUGGCAUUAAAACTT | 678 | ||
XM_168069 | CAAAGUUAUUAGCCCCAAGTT | 679 | ||
XM_168069 | CAGAGGCCAAGUAUAUCAATT | 680 | ||
XM_168069 | CCUGCAGAUUUGCACAGCGTT | 681 | ||
NM_021170 | AUCCUGGAGAUGACCGUGATT | 682 | ||
NM_021170 | GCCGGUCAUGGAGAAGCGGTT | 683 | ||
NM_021170 | UGGCCCUGAGACUGCAUCGTT | 684 | ||
NM_019089 | CCCCUCCAUGCUCAGAACUTT | 685 | ||
NM_019089 | CCUAUCUGGGAAGCCUGUGTT | 686 | ||
NM_019089 | UGCCCCAGUGACAAUAACATT | 687 | ||
| ACCAGGGCCAAAAUUAUGGTT | 688 | ||
NM_016653 | AUAGUGAACCUGGAACUGGTT | 689 | ||
NM_016653 | GCAGUUGCCCCAGAAGUUUTT | 690 | ||
NM_016281 | AGAACACACUGCUUGGUUGTT | 691 | ||
NM_016281 | GACAGUGAACAUGGAACCATT | 692 | ||
NM_016281 | GAGAACUUGCAGCACACACTT | 693 | ||
NM_012119 | ACCUGCCAACCUGCUCAUCTT | 694 | ||
NM_012119 | GAUCUCCUUUAAGGAGCAGTT | 695 | ||
NM_012119 | GCAGCUGUGUAUUUAAGGATT | 696 | ||
AB002301 | AGACAAAGAGGGGACCUUCTT | 697 | ||
AB002301 | GAAAGUCUAUCCGAAGGCUTT | 698 | ||
AB002301 | UGCCUCCCUGAAACUUCGATT | 699 | ||
NM_018401 | AGGUAUGCAUCGUGCAGAATT | 700 | ||
NM_018401 | GCAAUCAAACCGUCAUGACTT | 701 | ||
NM_018401 | UAUCCUGCUGGAUGAACACTT | 702 | ||
NM_006622 | GCAAGGUAUACAAUGCCGUTT | 703 | ||
NM_006622 | UAACUCAGCAACCCAGCAATT | 704 | ||
NM_006622 | UGCCUUGAAGACAGUACCATT | 705 | ||
AI278633 | CCUCAGCCGUAUAAUACGUTT | 706 | ||
AI278633 | CUGCUCUGUUCAAUCCCAGTT | 707 | ||
AI278633 | CUGGGAUUGGCCACCUCUUTT | 708 | ||
NM_152524 | AGAAGGAGAGUGUCAGGUUTT | 709 | ||
NM_152524 | UAUGUACCCCGUUCAGCAATT | 710 | ||
NM_152524 | UUUUGCCUUGGAGUGCUCCTT | 711 | ||
NM_019013 | AAAACCCCCGGGAGUCGUCTT | 712 | ||
NM_019013 | AGUGGCACCAAGUGGCUGGTT | 713 | ||
NM_019013 | GAAACCUGCUUUGUCAUUUTT | 714 | ||
AI338451 | CUGAUGCACUUUGCUGCAGTT | 715 | ||
AI338451 | CUGCAGGUUCAAAUCCCAGTT | 716 | ||
AI338451 | GGGGAAAAAGCUUUGCGUUTT | 717 | ||
NM_018410 | AAAGACCCAGGCUAUCAGATT | 718 | ||
NM_018410 | CAGACCCCAAAUCCAUAAGTT | 719 | ||
NM_018410 | GUCAGUGUCACCCAGCAAATT | 720 | ||
NM_018123 | UAUCGAGCCACCAUUUGUGTT | 721 | ||
NM_018123 | UGAUGCAUAUAGCCGCAACTT | 722 | ||
NM_018123 | UGCACAGGGCCAAAGUUGATT | 723 |
Table III C is used for the siRNA sequence of the screening of DNA disrupting agent: the camptothecine screening
Gene symbol | Serial ID | Adopted sequence is arranged | SEQ?ID?NO |
AATK | AB014541 | CGCAAGAAGAAGGCCGUGUTT | 724 |
AATK | AB014541 | CGCUGGUGCAAUGUUUUCUTT | 725 |
AATK | AB014541 | GAAUCCCUACCGAGACUCUTT | 726 |
ABL1 | NM_007313 | AAACCUCUACACGUUCUGCTT | 727 |
ABL1 | NM_007313 | CUAAAGGUGAAAAGCUCCGTT | 728 |
ABL1 | NM_007313 | UCCUGGCAAGAAAGCUUGATT | 729 |
ABL2 | NM_007314 | AUCAGUGAUGUGGUGCAGATT | 730 |
ABL2 | NM_007314 | GACUCGGACACUGAAGAAATT | 731 |
ABL2 | NM_007314 | UGGCACAGCAGGUACUAAATT | 732 |
ACVR2 | NM_001616 | AAGAUGGCCACAAACCUGCTT | 733 |
ACVR2 | NM_001616 | AGAUAAACGGCGGCAUUGUTT | 734 |
ACVR2 | NM_001616 | GACAUGCAGGAAGUUGUUGTT | 735 |
ACVR2B | NM_001106 | CGGGAGAUCUUCAGCACACTT | 736 |
ACVR2B | NM_001106 | GAGAUUGGCCAGCACCCUUTT | 737 |
ACVR2B | NM_001106 | GCCCAGGACAUGAGUGUCUTT | 738 |
AKT2 | NM_001626 | AGAUGGCCACAUCAAGAUCTT | 739 |
AKT2 | NM_001626 | GUCAUCAUUGCCAAGGAUGTT | 740 |
AKT2 | NM_001626 | UGCCAGCUGAUGAAGACCGTT | 741 |
ANAPC5 | NM_016237 | ACAGUGCUGAACUUGGCUUTT | 742 |
ANAPC5 | NM_016237 | CCAAAUGUCAGAGGCACAUTT | 743 |
ANAPC5 | NM_016237 | UCAAACUGAUGGCUGAAGGTT | 744 |
AXIN1 | AF009674 | GAAAGUGAGCGACGAGUUUTT | 745 |
AXIN1 | AF009674 | GUGCCUUCAACACAGCUUGTT | 746 |
AXIN1 | AF009674 | UGAAUAUCCAAGAGCAGGGTT | 747 |
BCL2 | NM_000633 | AGGACAUUUGUUGGAGGGGTT | 748 |
BCL2 | NM_000633 | GCUACCAAUUGUGCCGAGATT | 749 |
BCL2 | NM_000633 | UGAAGAACGUGGACGCUUUTT | 750 |
BLK | NM_001715 | AGUCACGAGCGUUCGAAAATT | 751 |
BLK | NM_001715 | CAACAUGAAGGUGGCCAUUTT | 752 |
BLK | NM_001715 | GCACUAUAAGAUCCGCUGCTT | 753 |
BMPR1B | NM_001203 | ACAGAUUGGAAAAGGUCGCTT | 754 |
BMPR1B | NM_001203 | GAAGUUACGCCCCUCAUUCTT | 755 |
BMPR1B | NM_001203 | UAUUUGCAGCACAGACGGATT | 756 |
BMPR2 | NM_001204 | CAAAUCUGUGAGCCCAACATT | 757 |
BMPR2 | NM_001204 | CAAGAUGUUCUUGCACAGGTT | 758 |
BMPR2 | NM_001204 | GAACGGCUAUGUGCGUUUATT | 759 |
BRCA1 | NM_007296 | ACUUAGGUGAAGCAGCAUCTT | 760 |
BRCA1 | NM_007296 | GGGCAGUGAAGACUUGAUUTT | 761 |
BRCA1 | NM_007296 | UGAAGUGGGCUCCAGUAUUTT | 762 |
BRCA2 | NM_000059 | CAAAUGGGCAGGACUCUUATT | 763 |
BRCA2 | NM_000059 | CUGUUCAGCCCAGUUUGAATT | 764 |
BRCA2 | NM_000059 | UCUCCAAGGAAGUUGUACCTT | 765 |
C20orf97 | NM_021158 | AGUCCCAGGUGGGACUCUUTT | 766 |
C20orf97 | NM_021158 | CUGGCAUCCUUGAGCUGACTT | 767 |
C20orf97 | NM_021158 | GACUGUUCUGGAAUGAGGGTT | 768 |
CAMK2D | NM_001221 | ACCAGAUGGAGUAAAGGAGTT | 769 |
CAMK2D | NM_001221 | GCACCCUAAUAUUGUGCGATT | 770 |
CAMK2D | NM_001221 | UUGGCAGACUUUGGCUUAGTT | 771 |
CCND1 | NM_053056 | CAUGUAACCGGCAUGUUUCTT | 772 |
CCND1 | NM_053056 | CCCACAGCUACUUGGUUUGTT | 773 |
CCND1 | NM_053056 | UGACCCCGCACGAUUUCAUTT | 774 |
CCNE2 | NM_057749 | CCACAGAUGAGGUCCAUACTT | 775 |
CCNE2 | NM_057749 | CUGGGGCUUUCUUGACAUGTT | 776 |
CCNE2 | NM_057749 | GUGGUUAAGAAAGCCUCAGTT | 777 |
CCNT2 | NM_058241 | AGCGCCAGUAAAGAAGAACTT | 778 |
CCNT2 | NM_058241 | AGGGCAGCCAGUUGUCAUUTT | 779 |
CCNT2 | NM_058241 | CCACCACUCCAAAAUGAGCTT | 780 |
CDC14B | NM_033331 | GGCCAUCCCCUCCAUUAAUTT | 781 |
CDC14B | NM_033331 | GUAAUUGAAAGGCAGUGCCTT | 782 |
CDC14B | NM_033331 | UUGCUAUCACUGUGGCUCUTT | 783 |
CDC16 | NM_003903 | AGUGGCUUCAAAGAUCCCUTT | 784 |
CDC16 | NM_003903 | GCAUGUCGUUACCUUGCAGTT | 785 |
CDC16 | NM_003903 | UAAGCCUAGUGAAACGGUCTT | 786 |
CDC23 | NM_004661 | AGCAACUGCUGCUUAUUGCTT | 787 |
CDC23 | NM_004661 | AGCAAGCAAGGAGAUAGGATT | 788 |
CDC23 | NM_004661 | CCUUCUUUAUGUCAGGAGCTT | 789 |
CDC25B | NM_021874 | AGGAUGAUGAUGCAGUUCCTT | 790 |
CDC25B | NM_021874 | GACAAGGAGAAUGUGCGCUTT | 791 |
CDC25B | NM_021874 | GAGCCCAGUCUGUUGAGUUTT | 792 |
CDC34 | NM_004359 | ACGUGGACGCCUCCGUGAUTT | 793 |
CDC34 | NM_004359 | CACCUACUACGAGGGCGGCTT | 794 |
CDC34 | NM_004359 | CAUCUACGAGACGGGGGACTT | 795 |
CDC37 | NM_007065 | CGCAUGGAGCAGUUCCAGATT | 796 |
CDC37 | NM_007065 | GACGGCUUCAGCAAGAGCATT | 797 |
CDC37 | NM_007065 | GAUUAAGACAGCCGAUCGCTT | 798 |
CDC42 | NM_044472 | ACCUUAUGGAAAAGGGGUGTT | 799 |
CDC42 | NM_044472 | CCAUCCUGUUUGAAAGCCUTT | 800 |
CDC42 | NM_044472 | CCCAAAAGGAAGUGCUGUATT | 801 |
CDC45L | NM_003504 | CACCUGCUCAAGUCCUUUGTT | 802 |
CDC45L | NM_003504 | GAUCCUUCAGGCCUUGUUCTT | 803 |
CDC45L | NM_003504 | UGACAGUGAUGGGUCAGAGTT | 804 |
CDK2 | NM_001798 | AUGAUAGCGGGGGCUAAGUTT | 805 |
CDK2 | NM_001798 | GAGCUAUCUGUUCCAGCUGTT | 806 |
CDK2 | NM_001798 | UCUAUUGCUUCACCAUGGCTT | 807 |
CDK2AP1 | NM_004642 | AGCAAAUACGCGGAGCUGCTT | 808 |
CDK2AP1 | NM_004642 | CUGCCCAGGUUUUUUUUGUTT | 809 |
CDK2AP1 | NM_004642 | GUUACAGUUCAUCUCCCCUTT | 810 |
CDK4 | NM_000075 | CCCUGGUGUUUGAGCAUGUTT | 811 |
CDK4 | NM_000075 | CUGACCGGGAGAUCAAGGUTT | 812 |
CDK4 | NM_000075 | GAGUGUGAGAGUCCCCAAUTT | 813 |
CDK5R2 | NM_003936 | AGGCGAGAGCCGACUCAAGTT | 814 |
CDK5R2 | NM_003936 | CCUGGACCGCUAGGGAUACTT | 815 |
CDK5R2 | NM_003936 | CGCAACCGCGAGAACCUUCTT | 816 |
CDK7 | NM_001799 | AACUGGCAGAUUUUGGCCUTT | 817 |
CDK7 | NM_001799 | CUGUCCAGUGGAAACCUUATT | 818 |
CDK7 | NM_001799 | UAGAACCGCCUUAAGAGAGTT | 819 |
CDKL5 | NM_003159 | ACAGUACCCAAUUCCGACATT | 820 |
CDKL5 | NM_003159 | GGAGAAUACUUCUGCUGUGTT | 821 |
CDKL5 | NM_003159 | UCAGCCACAAUGAUGUCCUTT | 822 |
CDKN1A | NM_078467 | AACUAGGCGGUUGAAUGAGTT | 823 |
CDKN1A | NM_078467 | CAUACUGGCCUGGACUGUUTT | 824 |
CDKN1A | NM_078467 | GAUGGUGGCAGUAGAGGCUTT | 825 |
CHEK1 | NM_001274 | AUCGAUUCUGCUCCUCUAGTT | 826 |
CHEK1 | NM_001274 | CUGAAGAAGCAGUCGCAGUTT | 827 |
CHEK1 | NM_001274 | UGCCUGAAAGAGACUUGUGTT | 828 |
CHEK1 | NM_001274 | CCAGUUGAUGUUUGGUCCUTT | 829 |
CHEK1 | NM_001274 | UCUCAGACUUUGGCUUGGCTT | 830 |
CHEK1 | NM_001274 | UUCUAUGGUCACAGGAGAGTT | 831 |
CHFR | NM_018223 | AGACUGCGUCCUUUUCGUCTT | 832 |
CHFR | NM_018223 | GAUACCAGCACCAGUGGAATT | 833 |
CHFR | NM_018223 | GCAUACCUCAUCCAGCAUCTT | 834 |
CKAP2 | NM_018204 | CCAAUCACAAGUCCUAUUGTT | 835 |
CKAP2 | NM_018204 | CUUGUGCGACCUCCUAUUATT | 836 |
CKAP2 | NM_018204 | GAGAGAAAAGCUCGUCUGATT | 837 |
CREBBP | NM_004380 | AUUUUUGCGGCGCCAGAAUTT | 838 |
CREBBP | NM_004380 | GAAAAACGGAGGUCGCGUUTT | 839 |
CREBBP | NM_004380 | GAAAACAAAUGCCCCGUGCTT | 840 |
CSF1R | NM_005211 | AGUGCAGAAAGUCAUCCCATT | 841 |
CSF1R | NM_005211 | CAACCUGCAGUUUGGUAAGTT | 842 |
CSF1R | NM_005211 | UGAGCCAAGUGGCAGCUAATT | 843 |
CTNNA1 | NM_001903 | CGUUCCGAUCCUCUAUACUTT | 844 |
CTNNA1 | NM_001903 | UGACAUCAUUGUGCUGGCCTT | 845 |
CTNNA1 | NM_001903 | UGACCAAAGAUGACCUGUGTT | 846 |
CTNNAL1 | NM_003798 | AAGUGUUGUUGCUGGCAGATT | 847 |
CTNNAL1 | NM_003798 | ACUUGAGAAGCUUUUGGGGTT | 848 |
CTNNAL1 | NM_003798 | CUAGAGGUUUUUGCUGCAGTT | 849 |
CTNNBIP1 | NM_020248 | AAAUUUGCGCCUCGGUAUCTT | 850 |
CTNNBIP1 | NM_020248 | ACCUAAGUCCUUCCACCUGTT | 851 |
CTNNBIP1 | NM_020248 | CACCCUGGAUGCUGUUGAATT | 852 |
CUL1 | NM_003592 | GACCGCAAACUACUGAUUCTT | 853 |
CUL1 | NM_003592 | GCCAGCAUGAUCUCCAAGUTT | 854 |
CUL1 | NM_003592 | UAGACAUUGGGUUCGCCGUTT | 855 |
DAPK2 | NM_014326 | GAAUAUUUUUGGGACGCCGTT | 856 |
DAPK2 | NM_014326 | UCCAAGAGGCUCUCAGACATT | 857 |
DAPK2 | NM_014326 | UCUCAGAAGGUCCUCCUGATT | 858 |
DCC | NM_005215 | ACAUCGUGGUGCGAGGUUATT | 859 |
DCC | NM_005215 | AUGAGCCGCCAAUUGGACATT | 860 |
DCC | NM_005215 | AUGGCAAGUUUGGAAGGACTT | 861 |
DDR1 | NM_013994 | AACAAGAGGACACAAUGGCTT | 862 |
DDR1 | NM_013994 | AGAGGUGAAGAUCAUGUCGTT | 863 |
DDR1 | NM_013994 | UCGCAGACUUUGGCAUGAGTT | 864 |
DMPK | NM_004409 | CAAGUGGGACAUGCUGAAGTT | 865 |
DMPK | NM_004409 | UAAAAGGCCCUCCAUCUGCTT | 866 |
DMPK | NM_004409 | UUGGCCCUGUUCAGCAAUGTT | 867 |
DTX1 | NM_004416 | AACCCACCUGAUGAGGACUTT | 868 |
DTX1 | NM_004416 | GACCGAGUUUGGAUCCAACTT | 869 |
DTX1 | NM_004416 | GAUGGAGUUCCACCUCAUCTT | 870 |
DYRK3 | NM_003582 | CCAUGUUUGCAUGGCCUUUTT | 871 |
DYRK3 | NM_003582 | CUUCUGGAGCAAUCCAAACTT | 872 |
DYRK3 | NM_003582 | UCUUUGGAUGCCCUCCACATT | 873 |
ECT2 | NM_018098 | ACUGGCUAAAGAUGCUGUGTT | 874 |
ECT2 | NM_018098 | GACCAUGGGAAAAUUGUGGTT | 875 |
ECT2 | NM_018098 | GCUUAGUACAGCGGGUUGATT | 876 |
EGR2 | NM_000399 | CACUACCACCCUUUCCUGUTT | 877 |
EGR2 | NM_000399 | GUGCAAUGUGAUGGGAGGATT | 878 |
EGR2 | NM_000399 | UGUUACCGGAGCUGAUUUGTT | 879 |
ELK1 | NM_005229 | GCCAUUCCUUUGUCUGCCATT | 880 |
ELK1 | NM_005229 | GUGAAAGUAGAAGGGCCCATT | 881 |
ELK1 | NM_005229 | UUCAAGCUGGUGGAUGCAGTT | 882 |
ELK1 | NM_005229 | AGGACCCUUUCAAUGUCCCTT | 883 |
ELK1 | NM_005229 | CUCUCAUUAUCUCCUCCACTT | 884 |
ELK1 | NM_005229 | GCUCUCCUUCCAGUUUCCATT | 885 |
EPHA4 | NM_004438 | CUGGCUACGAACUGAUUGGTT | 886 |
EPHA4 | NM_004438 | GAUUCCUAUCCGGUGGACUTT | 887 |
EPHA4 | NM_004438 | GCUAUCGUAUAGUUCGGACTT | 888 |
EPHB3 | NM_004443 | GAAGAUCCUGAGCAGUAUCTT | 889 |
EPHB3 | NM_004443 | GCUGCAGCAGUACAUUGCUTT | 890 |
EPHB3 | NM_004443 | UACCCUGGACAAGCUCAUCTT | 891 |
ETS1 | NM_005238 | UUCAGCCUGAAAGGUGUAGTT | 892 |
ETS1 | NM_005238 | ACGCUACGUGUACCGCUUUTT | 893 |
ETS1 | NM_005238 | UGACUACCCCUCGGUCAUUTT | 894 |
FLT1 | NM_002019 | ACAUCGAAAACAGCAGGUGTT | 895 |
FLT1 | NM_002019 | AGGAGGAGUGCAUCUUUGGTT | 896 |
FLT1 | NM_002019 | UGGAUGAGGACUUUUGCAGTT | 897 |
FOXO1A | NM_002015 | CUAUGCGUACUGCAUAGCATT | 898 |
FOXO1A | NM_002015 | GACAACGACACAUAGCUGGTT | 899 |
FOXO1A | NM_002015 | UACAAGGAACCUCAGAGCCTT | 900 |
FRAT1 | NM_005479 | AAGCUAAUGACGAGGAACCTT | 901 |
FRAT1 | NM_005479 | CCAUGGUGAAGUGCUUGGATT | 902 |
FRAT1 | NM_005479 | UAACAGCUGCAAUUCCCUGTT | 903 |
FRK | NM_002031 | ACUAUAGACUUCCGCAACCTT | 904 |
FRK | NM_002031 | CAGUAGAUUGCUGUGGCCUTT | 905 |
FRK | NM_002031 | CUCCAUACAGCUUCUGAAGTT | 906 |
FZD9 | NM_003508 | GACUUUCCAGACCUGGCAGTT | 907 |
FZD9 | NM_003508 | GAUCGGGGUCUUCUCCAUCTT | 908 |
FZD9 | NM_003508 | GGACUUCGCGCUGGUCUGGTT | 909 |
GPRK6 | NM_002082 | AAGCAAGAAAUGGCGGCAGTT | 910 |
GPRK6 | NM_002082 | GAGCUGAAUGUCUUUGGGCTT | 911 |
GPRK6 | NM_002082 | UGUAUAUAGCGACCAGAGCTT | 912 |
GUK1 | NM_000858 | CGGCAAAGAUUACUACUUUTT | 913 |
GUK1 | NM_000858 | GGAGCCCGGCCUGUUUGAUTT | 914 |
GUK1 | NM_000858 | UCAAGAAAGCUCAAAGGACTT | 915 |
HDAC3 | NM_003883 | CCCAGAGAUUUUUGAGGGATT | 916 |
HDAC3 | NM_003883 | UGCCUUCAACGUAGGCGAUTT | 917 |
HDAC3 | NM_003883 | UGGUACCUAUUAGGGAUGGTT | 918 |
HDAC4 | NM_006037 | AGAGGACGUUUUCUACGGCTT | 919 |
HDAC4 | NM_006037 | AUCUGUUUGCAAGGGGAAGTT | 920 |
HDAC4 | NM_006037 | CAAGAUCAUCCCCAAGCCATT | 921 |
HDAC5 | NM_005474 | AAACUGUUCUCAGAUGCCCTT | 922 |
HDAC5 | NM_005474 | CCCAACUUGAAAGUGCGUUTT | 923 |
HDAC5 | NM_005474 | UGAGAUGCACUCCUCCAGUTT | 924 |
HDAC9 | NM_058176 | AAGCUUCUUGUAGCUGGUGTT | 925 |
HDAC9 | NM_058176 | AUAUUGCCUGGACAGGUGGTT | 926 |
HDAC9 | NM_058176 | CAGCAACAAGAACUCCUAGTT | 927 |
HSPCB | NM_007355 | AGCAUUCAUGGAGGCUCUUTT | 928 |
HSPCB | NM_007355 | AUUGACAUCAUCCCCAACCTT | 929 |
HSPCB | NM_007355 | CUCAGCUUUUGUGGAGCGATT | 930 |
IRS1 | NM_005544 | AGGGCAGUGGAGACUAUAUTT | 931 | |
IRS1 | NM_005544 | CCAGAGUGCCAAAGUGAUCTT | 932 | |
IRS1 | NM_005544 | GGAUAUAUUUGGCUGGGUGTT | 933 | |
KIF17 | XM_027915 | GAUAACGGCUUCUGGAAGATT | 934 | |
KIF17 | XM_027915 | GCAAAAGCAACUUUGGCAGTT | 935 | |
KIF17 | XM_027915 | GCUCAAUAUCAGCUGGGAATT | 936 | |
KIF25 | NM_005355 | GAGCUAUACCAUGCUGGGATT | 937 | |
KIF25 | NM_005355 | GGAUGGACGGACAGAGGUUTT | 938 | |
KIF25 | NM_005355 | GUUACUGGUGAUUCUCUGCTT | 939 | |
KIF26A | XM_050278 | AUGCGGAAUUUGCCGUGGGTT | 940 | |
KIF26A | XM_050278 | GCACAAGCACCUGUGUGAGTT | 941 | |
KIF26A | XM_050278 | GUCGUACACCAUGAUCGGGTT | 942 | |
KIF2C | NM_006845 | ACAAAAACGGAGAUCCGUCTT | 943 | |
KIF2C | NM_006845 | AUAAGCAGCAAGAAACGGCTT | 944 | |
KIF2C | NM_006845 | GAAUUUCGGGCUACUUUGGTT | 945 | |
KIF3B | NM_004798 | AAACGGUCCAUUGGUAGGATT | 946 | |
KIF3B | NM_004798 | AAGUGGAAGGAAGUCGGGATT | 947 | |
KIF3B | NM_004798 | UGCCAAGCAGUUUGAACUGTT | 948 | |
KIF4B | AF241316 | CCUGCAGCAACUGAUUACCTT | 949 | |
KIF4B | AF241316 | GAACUUGAGAAGAUGCGAGTT | 950 | |
KIF4B | AF241316 | GAAGAGGCCCACUGAAGUUTT | 951 | |
KRAS2 | NM_033360 | GAAAAGACUCCUGGCUGUGTT | 952 | |
KRAS2 | NM_033360 | GGACUCUGAAGAUGUACCUTT | 953 | |
KRAS2 | NM_033360 | GGCAUACUAGUACAAGUGGTT | 954 | |
LATS2 | NM_014572 | AACAGCCAUCCAAGUCUUCTT | 955 | |
LATS2 | NM_014572 | AACCUACCAGCAGAAGGUUTT | 956 | |
LATS2 | NM_014572 | UAGGCUUUUCAGGACCUUCTT | 957 | |
MAP2K7 | NM_005043 | AGUCCUACAGGAAGAGCCCTT | 958 | |
MAP2K7 | NM_005043 | GCUACUUGAACACAGCUUCTT | 959 | |
MAP2K7 | NM_005043 | UCAACGACCUGGAGAACUUTT | 960 | |
MAP3K1 | AF042838 | UCACUUAGCAGCUGAGUCUTT | 961 | |
MAP3K1 | AF042838 | UUGACAGCACUGGUCAGAGTT | 962 | |
MAP3K1 | AF042838 | UUGGCAAGAACUUCUUGGCTT | 963 | |
MAP3K4 | NM_005922 | AGAACGAUCGUCCAGUGGATT | 964 | |
MAP3K4 | NM_005922 | GGUACCUCGAUGCCAUAGUTT | 965 | |
MAP3K4 | NM_005922 | UUUUGGACUAGUGCGGAUGTT | 966 | |
MAP4K5 | NM_006575 | AAGGCUGCCACAAAUGUUGTT | 967 | |
MAP4K5 | NM_006575 | GAAACAGAAGCACGAGAUGTT | 968 | |
MAP4K5 | NM_006575 | UCUCUACAUCUUGGCUGGATT | 969 | |
MAPK13 | NM_002754 | CUCACAGUGGAUGAAUGGATT | 970 | |
MAPK13 | NM_002754 | GAUCAUGGGGAUGGAGUUCTT | 971 | |
MAPK13 | NM_002754 | UACAGCCUUUCAAGCAGAGTT | 972 | |
MAPK8 | NM_139049 | CACCCGUACAUCAAUGUCUTT | 973 | |
MAPK8 | NM_139049 | GGAAUAGUAUGCGCAGCUUTT | 974 | |
MAPK8 | NM_139049 | GUGAUUCAGAUGGAGCUAGTT | 975 | |
MAPRE1 | NM_012325 | GAGUAUUAACAGCCUGGACTT | 976 | |
MAPRE1 | NM_012325 | GCUAAGCUAGAACACGAGUTT | 977 | |
MAPRE1 | | UAGAGGAUGUGUUUCAGCCTT | 978 | |
MAPRE3 | NM_012326 | CAGCUUUGUUCAGGGGCAGTT | 979 | |
MAPRE3 | NM_012326 | CUUCGUGACAUCGAGCUCATT | 980 | |
MAPRE3 | NM_012326 | GGAUUACAACCCUCUGCUGTT | 981 | |
MARK1 | NM_018650 | ACAACAGCACUCUUCAGUCTT | 982 |
MARK1 | NM_018650 | CUGCGAGAGCGAGUUUUACTT | 983 |
MARK1 | NM_018650 | UGUGUAUUCUGGAGGUAGCTT | 984 |
MCC | NM_002387 | AGUUGAGGAGGUUUCUGCATT | 985 |
MCC | NM_002387 | GACUUAGAGCUGGGAAUCUTT | 986 |
MCC | NM_002387 | GGAUUAUAUCCAGCAGCUCTT | 987 |
MCM3 | NM_002388 | AGGAUUUUGUGGCCUCCAUTT | 988 |
MCM3 | NM_002388 | GUCUCAGCUUCUGCGGUAUTT | 989 |
MCM3 | NM_002388 | UCCAGGUUGAAGGCAUUCATT | 990 |
MCM3 | NM_002388 | GCAGAUGAGCAAGGAUGCUTT | 991 |
MCM3 | NM_002388 | GUACAUCCAUGUGGCCAAATT | 992 |
MCM3 | NM_002388 | UGGGUCAUGAAAGCUGCCATT | 993 |
MLH1 | NM_000249 | AACUGAAAGCCCCUCCUAATT | 994 |
MLH1 | NM_000249 | GAUGGAAAUAUCCUGCAGCTT | 995 |
MLH1 | NM_000249 | UGCUGUUAGUCGAGAACUGTT | 996 |
MYB | NM_005375 | ACAAGAGGUGGAAUCUCCATT | 997 |
MYB | NM_005375 | GGUUAUCUGCAGGAGUCUUTT | 998 |
MYB | NM_005375 | UCGAACAGAUGUGCAGUGCTT | 999 |
MYO3A | NM_017433 | AAAGCUACCGAUGUCAGGGTT | 1000 |
MYO3A | NM_017433 | AAAUCCCGAGUUAUCCACCTT | 1001 |
MYO3A | NM_017433 | GGCUAAUGAAAGGUGCUGGTT | 1002 |
NEK1 | AB067488 | AAGUGACAUUUGGGCUCUGTT | 1003 |
NEK1 | AB067488 | AUGCACGUGCUGCUGUACUTT | 1004 |
NEK1 | AB067488 | GAAGGACCUUCUGAUUCUGTT | 1005 |
NF1 | NM_000267 | AUCCUUCAACAAGGCACAGTT | 1006 |
NF1 | NM_000267 | GUAACUUCAGCAGAGCGAATT | 1007 |
NF1 | NM_000267 | UACAUGACUCCAUGGCUGUTT | 1008 |
NFKB2 | NM_002502 | AGGAUUCUCAUGGGAAGGGTT | 1009 |
NFKB2 | NM_002502 | GAAGAACAUGAUGGGGACUTT | 1010 |
NFKB2 | NM_002502 | GAUUGAGCGGCCUGUAACATT | 1011 |
NTRK1 | NM_002529 | CAACGGCAACUACACGCUGTT | 1012 |
NTRK1 | NM_002529 | CGCCACAGCAUCAAGGAUGTT | 1013 |
NTRK1 | NM_002529 | GAGUGGUCUCCGUUUCGUGTT | 1014 |
OSR1 | NM_005109 | GAUACACAAAGAUGGGCUGTT | 1015 |
OSR1 | NM_005109 | AAACAGCUCAGGCUUUGUCTT | 1016 |
OSR1 | NM_005109 | GAAUAGUGGCUUACCGCUUTT | 1017 |
PAK1 | NM_002576 | CCGCUGUCUCGAUAUGGAUTT | 1018 |
PAK1 | NM_002576 | GGACCGAUUUUACCGAUCCTT | 1019 |
PAK1 | NM_002576 | UGGAUGGCUCUGUCAAGCUTT | 1020 |
PCNA | NM_002592 | AAUUGCGGAUAUGGGACACTT | 1021 |
PCNA | NM_002592 | AGUCCAAAGUCUGAUCUGGTT | 1022 |
PCNA | NM_002592 | UUUCCUGUGCAAAAGACGGTT | 1023 |
PDGFRB | NM_002609 | AAAGAAGUACCAGCAGGUGTT | 1024 |
PDGFRB | NM_002609 | UCCAUCCACCAGAGUCUAGTT | 1025 |
PDGFRB | NM_002609 | UUUGCUGAGCUGCAUCGGATT | 1026 |
PDZGEF2 | NM_016340 | AACCCUCAUCCACAGGUGATT | 1027 |
PDZGEF2 | NM_016340 | CCGACUGAGUACAUCGAUGTT | 1028 |
PDZGEF2 | NM_016340 | GCCAGAUUCGACUGAUUGUTT | 1029 |
PIK3C2A | NM_002645 | AACGAGGAAUCCGACAUUCTT | 1030 |
PIK3C2A | NM_002645 | UGAUGAGCCCAUCCUUUCATT | 1031 |
PIK3C2A | NM_002645 | UGCUUCAACGGAUGUAGCATT | 1032 |
POLS | NM_006999 | ACAGAGACGCCGAAAGUACTT | 1033 |
POLS | NM_006999 | CUAGCGACAUAGACCUGGUTT | 1034 |
POLS | NM_006999 | GCAAAUGAAUUGGCCUGGCTT | 1035 |
PPARG | NM_015869 | AAUGACAGACCUCAGACAGTT | 1036 |
PPARG | NM_015869 | UAAGCCUCAUGAAGAGCCUTT | 1037 |
PPARG | NM_015869 | UGUCAGUACUGUCGGUUUCTT | 1038 |
PRC1 | NM_003981 | AAGCAUAUCCGUCUGUCAGTT | 1039 |
PRC1 | NM_003981 | AGGCUUCCAAAUCUGAUGCTT | 1040 |
PRC1 | NM_003981 | GGAACUCUUUGAAGGUGUCTT | 1041 |
PRKACA | NM_002730 | GAAUGGGGUCAACGAUAUCTT | 1042 |
PRKACA | NM_002730 | GGACGAGACUUCCUCUUGATT | 1043 |
PRKACA | NM_002730 | GUGUGGCAAGGAGUUUUCUTT | 1044 |
PRKCB1 | NM_002738 | AGAGCAUGCAUUUUUCCGGTT | 1045 |
PRKCB1 | NM_002738 | GGAGCCCCAUGCUGUAUUUTT | 1046 |
PRKCB1 | NM_002738 | UUGGAUGUUAGCGGUACUCTT | 1047 |
PRKCL1 | NM_002741 | CACAAGAUCGUCUACAGGGTT | 1048 |
PRKCL1 | NM_002741 | CACCAGUGAAGUCAGCACUTT | 1049 |
PRKCL1 | NM_002741 | GAUUUCAAGUUCCUGGCGGTT | 1050 |
PRKCM | NM_002742 | AAUGAAUGAGGAGGGUAGGTT | 1051 |
PRKCM | NM_002742 | CCUUCAUCACCCUGGUGUUTT | 1052 |
PRKCM | NM_002742 | GUUCCCUGAAUGUGGUUUCTT | 1053 |
PRKWNK3 | AJ409088 | ACCAAGCAGCCAGCUAUACTT | 1054 |
PRKWNK3 | AJ409088 | CUAAUGACAUCUGGGACCUTT | 1055 |
PRKWNK3 | AJ409088 | CUACGAAGGAAAACGUCAGTT | 1056 |
PRKY | NM_002760 | AGACAGUGAAGCUGGUUGUTT | 1057 |
PRKY | NM_002760 | GAAUUUCUGAGGACGAGCUTT | 1058 |
PRKY | NM_002760 | UCAGAUUUGGGCCAGAGUUTT | 1059 |
PTEN | NM_000314 | UGGAGGGGAAUGCUCAGAATT | 1060 |
PTEN | NM_000314 | AAGGCAGCUAAAGGAAGUGTT | 1061 |
PTEN | NM_000314 | UAAAGAUGGCACUUUCCCGTT | 1062 |
PTK6 | NM_005975 | AACACCCUCUGCAAAGUUGTT | 1063 |
PTK6 | NM_005975 | CCGUGGUUCUUUGGCUGCATT | 1064 |
PTK6 | NM_005975 | UCAGGCUUAUCCGAUGUGCTT | 1065 |
PTTG1 | NM_004219 | AACAGCCAAGCUUUUCUGCTT | 1066 |
PTTG1 | NM_004219 | GGCUUUGGGAACUGUCAACTT | 1067 |
PTTG1 | NM_004219 | UCUGUUGCAGUCUCCUUCATT | 1068 |
RALA | NM_005402 | AGACAGGUUUCUGUAGAAGTT | 1069 |
RALA | NM_005402 | GUCCAGAUCGAUAUCUUAGTT | 1070 |
RALA | NM_005402 | GUUUAGCCAAGAGAAUCAGTT | 1071 |
RALBP1 | NM_006788 | AAUGAAGAGGUCCAAGGGATT | 1072 |
RALBP1 | NM_006788 | AGGACCCGUGCAUCUUACUTT | 1073 |
RALBP1 | NM_006788 | GCUAAAAGACAGGAGUGUGTT | 1074 |
RAP1A | NM_002884 | CAGUGUAUGCUCGAAAUCCTT | 1075 |
RAP1A | NM_002884 | GAUGAGCGAGUAGUUGGCATT | 1076 |
RAP1A | NM_002884 | UUGGAAAGUGCCAGCAUUCTT | 1077 |
RASA2 | NM_006506 | AACUGAUGACCUGGGGUCUTT | 1078 |
RASA2 | NM_006506 | CAAGCAGAGAGCUCACCUATT | 1079 |
RASA2 | NM_006506 | GAAAACAAGCAAUCCGCAGTT | 1080 |
RET | NM_000323 | CUUCGCAGAAAAGAGUCGGTT | 1081 |
RET | NM_000323 | GACAUCCAGGAUCCACUGUTT | 1082 |
RET | NM_000323 | GUGUGCCGAACUUCACUACTT | 1083 |
RHOK | NM_002929 | AGUACACAGCAGGUUCAUCTT | 1084 |
RHOK | NM_002929 | CGUGAAUGAGGAGAACCCUTT | 1085 |
RHOK | NM_002929 | GUUUAAGGAGGGGCCUGUGTT | 1086 |
RPS6KA6 | NM_014496 | CCUCCUUUCAAACCUGCUUTT | 1087 |
RPS6KA6 | NM_014496 | GAGGUUCUGUUUACAGAGGTT | 1088 |
RPS6KA6 | NM_014496 | UCAGCCAGUGCAGAUUCAATT | 1089 |
SGK2 | NM_016276 | AGAGCCUUAUGAUCGAGCATT | 1090 |
SGK2 | NM_016276 | CUCUAUCAUGCCUGCUCCUTT | 1091 |
SGK2 | NM_016276 | GAGAAGGACCUGUGAAACUTT | 1092 |
SKP2 | NM_005983 | AAGAACCAGGAGAUAUGGGTT | 1093 |
SKP2 | NM_005983 | GGUCUCUGGUGUUUGUAAGTT | 1094 |
SKP2 | NM_005983 | UUUGCCCUGCAGACUUUGCTT | 1095 |
SRC | NM_005417 | GAACCGGAUGCAGUUGAGCTT | 1096 |
SRC | NM_005417 | GCCGGAAUACAAGAACGGGTT | 1097 |
SRC | NM_005417 | GUGGCUCUUAUCCGCAUGATT | 1098 |
SRPK1 | NM_003137 | CGCUUAUGGAACGUGAUACTT | 1099 |
SRPK1 | NM_003137 | GCAACAGAAUGGCAGCGAUTT | 1100 |
SRPK1 | NM_003137 | GUUCUAAUCGGAUCUGGCUTT | 1101 |
STAT3 | NM_139276 | AUGCCACAGGCCACCUAUATT | 1102 |
STAT3 | NM_139276 | CGACCUGCAGCAAUACCAUTT | 1103 |
STAT3 | NM_139276 | GAAUCACAUGCCACUUUGGTT | 1104 |
STAT4 | NM_003151 | ACACAGAUCUGCCUCUAUGTT | 1105 |
STAT4 | NM_003151 | CCCUACAAUAAAGGCCGGUTT | 1106 |
STAT4 | NM_003151 | UUAGGAAGGUCCUUCAGGGTT | 1107 |
STAT5A | NM_003152 | CCUGUGGAACCUGAAACCATT | 1108 |
STAT5A | NM_003152 | GUCUAUGAUGCUGUUGCCCTT | 1109 |
STAT5A | NM_003152 | UGAGAUGAUUCAGAAGGGGTT | 1110 |
STK4 | NM_006282 | CACCAUUUUGGAUGGCUCCTT | 1111 |
STK4 | NM_006282 | GGAAAACCAGAUCAACAGCTT | 1112 |
STK4 | NM_006282 | UUCUGGAUGGCUUGCCUCATT | 1113 |
STK6 | NM_003600 | ACAGUCUUAGGAAUCGUGCTT | 1114 |
STK6 | NM_003600 | GCACAAAAGCUUGUCUCCATT | 1115 |
STK6 | NM_003600 | UUGCAGAUUUUGGGUGGUCTT | 1116 |
TCF3 | M31523 | AAAGACCCCGUGUAAACCUTT | 1117 |
TCF3 | M31523 | ACCUCAAGGCCAGCUCAAUTT | 1118 |
TCF3 | M31523 | AUGGGGCAUUUUGUUGGGATT | 1119 |
TERT | NM_003219 | CACCAAGAAGUUCAUCUCCTT | 1120 |
TERT | NM_003219 | GAGUGUCUGGAGCAAGUUGTT | 1121 |
TERT | NM_003219 | GUUUGGAAGAACCCCACAUTT | 1122 |
TGFBR1 | NM_004612 | GACAUGAUUCAGCCACAGATT | 1123 |
TGFBR1 | NM_004612 | UUCCUCGAGAUAGGCCGUUTT | 1124 |
TGFBR1 | NM_004612 | UUUGGGAGGUCAGUUGUUCTT | 1125 |
TK2 | NM_004614 | GAUGCCAGAAGUGGACUAUTT | 1126 |
TK2 | NM_004614 | UACCUGGAAGCAAUUCACCTT | 1127 |
TK2 | NM_004614 | UUAUGCUGCAUUUGGCUGGTT | 1128 |
top2B | NM_001068 | ACAUUCCCUGGAGUGUACATT | 1129 |
top2B | NM_001068 | GAGGAUUUAGCGGCAUUUGTT | 1130 |
top2B | NM_001068 | GCUGCUGGACUGCAUAAAGTT | 1131 |
top3A | NM_004618 | GAUCCUCCCUGUCUAUGAGTT | 1132 |
top3A | NM_004618 | GCAAAGAAAUUGGACGAGGTT | 1133 |
top3A | NM_004618 | GGCGAAAACAUCGGGUUUGTT | 1134 |
top3B | NM_003935 | CAAAUGGGACAAAGUGGACTT | 1135 |
top3B | NM_003935 | CUUUGACCUGAAGGGCUCUTT | 1136 |
top3B | NM_003935 | UCCAGUCCUUCAAACCAGATT | 1137 |
WASL | NM_003941 | AAACAGGAGGUGUUGAAGCTT | 1138 |
WASL | NM_003941 | AAGUGGAGCAGAACAGUCGTT | 1139 |
WASL | NM_003941 | GGACAAUCCACAGAGAUCUTT | 1140 |
WEE1 | NM_003390 | AUCGGCUCUGGAGAAUUUGTT | 1141 |
WEE1 | NM_003390 | CAAGGAUCUCCAGUCCACATT | 1142 |
WEE1 | NM_003390 | UGUACCUGUGUGUCCAUCUTT | 1143 |
WISP1 | NM_003882 | AAAUGCCUGUCUCUAGCUGTT | 1144 |
WISP1 | NM_003882 | AUGGCCAGUUUUCUGGUAGTT | 1145 |
WISP1 | NM_003882 | CCUGGGCAUUGUUGAGGUUTT | 1146 |
WISP3 | NM_003880 | ACAGUUUUGUCACUGGCCCTT | 1147 |
WISP3 | NM_003880 | CAAAAUGGACUCCCUGCUCTT | 1148 |
WISP3 | NM_003880 | CCAGGGGAAAUCUGCAAUGTT | 1149 |
WNT1 | NM_005430 | ACGGCGUUUAUCUUCGCUATT | 1150 |
WNT1 | NM_005430 | CCCUCUUGCCAUCCUGAUGTT | 1151 |
WNT1 | NM_005430 | CUAUUUAUUGUGCUGGGUCTT | 1152 |
WNT2 | NM_003391 | AUUUGCCCGCGCAUUUGUGTT | 1153 |
WNT2 | NM_003391 | AACGGGCGAUUAUCUCUGGTT | 1154 |
WNT2 | NM_003391 | AGAAGAUGAAUGGUCUGGCTT | 1155 |
WT1 | NM_024426 | CACUGGCACACUGCUCUUATT | 1156 |
WT1 | NM_024426 | GACAAGAUACCGGUGCUUCTT | 1157 |
WT1 | NM_024426 | GACACCAAAGGAGACAUACTT | 1158 |
NM_017719 | AGACCUAAUCACACGGAUGTT | 1159 | |
NM_017719 | AGAUAGCGGGUUCACCUACTT | 1160 | |
NM_017719 | GUUGACAGACUUUGGGUUCTT | 1161 | |
XM_168069 | ACUCCAUCUGGUUGACCUGTT | 1162 | |
XM_168069 | GAUUCAGGUGGAACUGAACTT | 1163 | |
XM_168069 | GCACCAAGCUCCUCUGAUGTT | 1164 | |
XM_170783 | ACCGACACUUUGGCUUCCATT | 1165 | |
XM_170783 | GAUGAGCGCGGGAAUGUUGTT | 1166 | |
XM_170783 | UGGCCGAGGCCUUCAAGCUTT | 1167 | |
XM_064050 | CAUCAAUCACUCUCUGCUGTT | 1168 | |
XM_064050 | CUAACCCAGGAUGUUCAGGTT | 1169 | |
XM_064050 | GACACUCACCAUGCUGAAATT | 1170 | |
XM_066649 | AAGGGUGACUUUGUGUCCUTT | 1171 | |
XM_066649 | ACCAGGAACAAACCUGUUGTT | 1172 | |
XM_066649 | UUUGAAGGUGGCCCUCCUATT | 1173 | |
XM_089006 | AAAUCGAGAAGGAGGCUCATT | 1174 | |
XM_089006 | AUAGUGACCGUCCCUUUGATT | 1175 | |
XM_089006 | CCAGGUUCCUCCAAAGAUGTT | 1176 | |
NM_145754 | AAGGGUUCAGCAUCUGACUTT | 1177 | |
NM_145754 | CCUGGAGACAUUGCACCAGTT | 1178 | |
NM_145754 | GGUGCUACCUCCUUUCCAGTT | 1179 | |
NM_017596 | AGUUGCCCACCCUGUUUUUTT | 1180 | |
NM_017596 | GAAAGAAUCCGUCCGCAUGTT | 1181 | |
NM_017596 | GCAGCCAGAACUCUCAAAGTT | 1182 | |
NM_139286 | GUUGUAGGAGGCAGUGAUGTT | 1183 | |
NM_139286 | UCUCAAUUUGGAAGUCUUGTT | 1184 | |
NM_139286 | UGAUCAAUGAUCGGAUUGGTT | 1185 | |
NM_014885 | ACCAGGAUUUGGAGUGGAUTT | 1186 | |
NM_014885 | CAAGGCAUCCGUUAUAUCUTT | 1187 | |
NM_014885 | GUGGCUGGAUUCAUGUUCCTT | 1188 | |
NM_016263 | CCAGAUCCUUGUCUGGAAGTT | 1189 | |
NM_016263 | CGACAACAAGCUGCUGGUCTT | 1190 |
NM_016263 | GAAGCUGUCCAUGUUGGAGTT | 1191 | |
NM_013367 | AGCCAGCAGAUGUAAUUGGTT | 1192 | |
NM_013367 | CAUUUCAAUGAGGCUCCAGTT | 1193 | |
NM_013367 | GUCAUUUACAGAGUGGCUCTT | 1194 | |
NM_018492 | AGGACACUUUGGGUACCAGTT | 1195 | |
NM_018492 | GACCCUAAAGAUCGUCCUUTT | 1196 | |
NM_018492 | GCUGAGGAGAAUAUGCCUCTT | 1197 | |
XM_168069 | CAAAGUUAUUAGCCCCAAGTT | 1198 | |
XM_168069 | CAGAGGCCAAGUAUAUCAATT | 1199 | |
XM_168069 | CCUGCAGAUUUGCACAGCGTT | 1200 | |
NM_021170 | AUCCUGGAGAUGACCGUGATT | 1201 | |
NM_021170 | GCCGGUCAUGGAGAAGCGGTT | 1202 | |
NM_021170 | UGGCCCUGAGACUGCAUCGTT | 1203 | |
NM_019089 | CCCCUCCAUGCUCAGAACUTT | 1204 | |
NM_019089 | CCUAUCUGGGAAGCCUGUGTT | 1205 | |
NM_019089 | UGCCCCAGUGACAAUAACATT | 1206 | |
AK024504 | AGAGAGCUGGACCAUUCAUTT | 1207 | |
AK024504 | AUGAGCAAUGCGGAUAGCUTT | 1208 | |
AK024504 | GCCAUGUGUCUGAUGACAUTT | 1209 | |
AB002301 | AGACAAAGAGGGGACCUUCTT | 1210 | |
AB002301 | GAAAGUCUAUCCGAAGGCUTT | 1211 | |
AB002301 | UGCCUCCCUGAAACUUCGATT | 1212 | |
NM_018401 | AGGUAUGCAUCGUGCAGAATT | 1213 | |
NM_018401 | GCAAUCAAACCGUCAUGACTT | 1214 | |
NM_018401 | UAUCCUGCUGGAUGAACACTT | 1215 | |
NM_016457 | CAUUGUCCACUGUGACUUGTT | 1216 | |
NM_016457 | UGAAGUGGCCAUUCUGCAGTT | 1217 | |
NM_016457 | UGUGGACAUUGCCACUGUCTT | 1218 | |
NM_005200 | AUGAUCGCACCGCAGAGGUTT | 1219 | |
NM_005200 | UACAUGACGUACUUGAGUGTT | 1220 | |
NM_005200 | UGCUAAGGGGAUCGGACAUTT | 1221 | |
NM_024322 | ACCACUCCGGAUACAUCACTT | 1222 | |
NM_024322 | ACUAAGGCGUCUGCGAGAUTT | 1223 | |
NM_024322 | GGACCUCACAGCAACUCUUTT | 1224 | |
NM_017769 | CUGGUUGCAGUUCCAUUCCTT | 1225 | |
NM_017769 | GUGAGCAUCCUGGAUCAAATT | 1226 | |
NM_017769 | UUCAGAGAGUCCACACACCTT | 1227 | |
NM_019013 | AAAACCCCCGGGAGUCGUCTT | 1228 | |
NM_019013 | AGUGGCACCAAGUGGCUGGTT | 1229 | |
NM_019013 | GAAACCUGCUUUGUCAUUUTT | 1230 | |
AI338451 | CUGAUGCACUUUGCUGCAGTT | 1231 | |
AI338451 | CUGCAGGUUCAAAUCCCAGTT | 1232 | |
AI338451 | GGGGAAAAAGCUUUGCGUUTT | 1233 | |
NM_018123 | UAUCGAGCCACCAUUUGUGTT | 1234 | |
NM_018123 | UGAUGCAUAUAGCCGCAACTT | 1235 | |
NM_018123 | UGCACAGGGCCAAAGUUGATT | 1236 |
6.4. embodiment 4: as the BRCA1/BARD1E3 ubiquitin ligase enzyme of cancer therapy drug target
The remarkable part of these discoveries is the product and holoenzyme mixture (BRCC) association (Dong et al., the Mol Cell.2003Nov that strengthen the cell survival after DNA destroys of BRCA1, BRCA2, BARD21 and RAD51 gene; 12 (5): 1087-99).This mixture has E3Ub ligase enzyme activity, and great majority wherein can be used as the BRCAl/BARD1 heterodimer and reclaim (Dong et al., Mol Cell.2003Nov; 12 (5): 1087-99; Brzovicet al., Nat Struct Biol.2001Oct; 8 (10): 833-7).These find to show strongly that BRCC participates in the susceptibility of mediation to cis-platinum in our siRNA screening.Unexpectedly, the member's (FANCA, FANCC, FANCE and FANCF) of another kind of many subunits mixture-FANC mixture siRNA set participates in resistance (Taniguchiet al., the Nat Med.2003May of decision to cis-platinum; 9 (5): 568-74), it does not increase susceptibility (data not shown goes out) in our mensuration.
In order to determine that BRCA1 or BRCA2 destroy the sensitization to cis-platinum that causes and whether be subjected to that TP53 expresses the influence that exists or lack in the target cell, used by the stably express target and decide the TP53 positive of the coupling that the short hairpin RNA (shRNA) of TP53 produces and negative cells (referring to embodiment 2).Dye TP53 positive or negative cell with the siRNA of BRCA1 or BRCA2 set excess revolutions, use cisplatin treated, and with Alamar Blue analysis of cells grow (Figure 20).When with BRCA1 or BRCA2siRNAs (the about 0.1nM of IC50) transfection, sensitivity was about 10 times when the TP53 negative cells was used contrast siRNA (luciferase, the about 1nM of IC50) transfection to the cis-platinum ratio.In lower cis-platinum concentration, BRCA1 or BRCA2 destroy the back to the sensibilized of cis-platinum even more remarkable.After BRCA1 or BRCA2 destroyed, the TP53 positive cell was to cis-platinum more insensitive (the about 0.4nM of IC50).In this is analyzed, BRCA1 or BRCA2 destroy the back to the sensibilized of cis-platinum on the order of magnitude with sensibilized similar (data not shown goes out) after CHEK1 destruction.Also can use cell cycle analysis, research BRCA1 and BRCA2 destroy the sensibilized of back to the DNA disrupting agent.The TP53 positive and negative cells are dyed in siRNA set excess revolutions with BRCA1 or BRCA2, with a kind of processing in some DNA disrupting agents (cis-platinum, camptothecine, Zorubicin and bleomycin), and by flow cytometry cell cycle destruction.In all cases, the TP53 negative cells BRCA1 and BRCA2 destroy the back than in the luciferase cells transfected to DNA disrupting agent more responsive (data not shown goes out).These cells were shown in Figure 21 to the reaction of bleomycin after BRCA1 destroyed.BRCA1 destroys after handling the TP53 negative cells with bleomycin, has caused more inferior G1 cell (dead cell) than handling the TP53 positive cell.Described result shows that the cell that lacks TP53 destroys the back at BRCA1 and destroys more responsive to DNA.Figure 22 represents to prove RAD51/ Zorubicin synergy stronger result in the TP53-cell.
The clone of using in the present embodiment is positive A549 cell of HeLa cell, TP53 and the negative A549 cell of TP53.Decide the short hairpin RNA (shRNA) of TP53 by the stable transfection target, prepared the positive and negative cells of the TP53 of coupling to (monthly highlthighlight, Nov.2003).With the siRNA of 100nM (every kind of siRNA 33nM) set (set of three kinds of siRNA of each gene) transfectional cell, or with the single siRNA transfectional cell of 100nM.Following siRNA:Luc contrast, BRCA1, BRCA2 and BARD1 set have been used in our research.Handle cells transfected with the DNA disrupting agent of various concentration then.The concentration of the every kind of reagent that uses in the cell cycle analysis is as follows: for the HeLa cell, use Zorubicin (10nM), camptothecine (6nM), cis-platinum (400ng/ml), ametycin (40nM), bleomycin (100ng/ml); For other clone, use Zorubicin (200nM), camptothecine (200nM), cis-platinum (2ug/ml) ametycin (400nM), bleomycin (5ug/ml).
Be performed as follows the siRNA transfection: in transfection the day before yesterday, the clone that 2000 (or 100) microlitre is selected, as at DMEM/10% foetal calf serum (Invitrogen, Carlsbad, CA) grow to the about 90% cervical cancer HeLa cell (ATCC that converges in, Cat.No.CCL-2) be planted in the tissue culturing plate of 6 holes (or 96 holes) with 45,000 (or 2000) cells/well.For each transfection, with 70 microlitre OptiMEM (Invitrogen) with (Dharmacon, Lafayette CO) mix from 5 microlitre siRNA of 20 micromole's liquid storages.For each transfection, 20 microlitre OptiMEM are mixed with 5 microlitre Oligofectamine reagent (Invitrogen), and incubation 5 minutes at room temperature.Then 25 microlitre OptiMEM/Oligofectamine mixtures are mixed with 75 microlitre OptiMEM/siRNA mixtures, and at room temperature incubation 15-20 minute.100 (or 10) microlitre transfection mixture is distributed in each hole of 6 holes (or 96 holes) plate, and under the condition of 37 ℃ and 5%CO2 incubation 4 hours.
After 4 hours, the DMEM/10% foetal calf serum that contains or do not contain the DNA disrupting agent in 100 microlitres/hole is added each hole, to reach the final concentration of every kind of reagent mentioned above.With plate incubation 68 hours again under the condition of 37 ℃ and 5%CO2.Analysis is from the cell cycle spectrum of the sample of 6 orifice plates, with the cell growth of Alamar Blue assay method analysis from the sample of 96 orifice plates.
For cell cycle analysis, will be from the supernatant liquor and the cytomixis of gathering in the crops by tryptic digestion in each hole.Then with 1200rpm with centrifugal 5 minutes of mixture.With 70% ice-cold ethanol with about 30 minutes of cell fixation.With PBS once, be suspended in 0.5ml again and contain iodate third ingot (among the PBS of 10 micrograms/ml) and RNA enzyme A (1mg/ml), and 37 ℃ of following incubations 30 minutes with the fixed cell washing.(Becton Dickinson) carries out flow cytometry with the FACSCalibur flow cytometer, and with FlowJo software (TreeStar, Inc) analytical data.Measure necrocytosis with inferior G1 cell mass.If (siRNA+DMSO) summation of the inferior G1 cell mass of sample and (Luc+ medicine) sample is greater than the inferior G1 cell mass of (siRNA+ medicine) sample, we determine that the siRNA silence is to the sensitization of DNA destructive.
Analyze for Alamar Blue, remove the substratum of 96 orifice plates, add the 100uL/ hole contain 10% (vol/vol) alamar Blue reagent (BioSource International, Inc) and the perfect medium of 1 percent volume 1M Hepes damping fluid tissue culture reagent.Then with plate at 37 ℃ of following incubation 1-4 hours, by exciting and detect emission at 590nm and measure fluorescence at 544nm with SPECTRAMax Gemini-Xs spectrofluorimeter (Molceular Devices).Background (acellular) is proofreaied and correct fluorescent signal.Cell response (survival) under the DNA disrupting agent exists is measured as the per-cent of the control cells growth that does not exist under the DNA disrupting agent condition.
BRCA1 has a lot of functions, but unique known enzyme function is an E3Ub ligase enzyme activity.This activity strengthens by the association of BARD1 and BRCA1, and causes the BRCA1/BARD1 mixture by unconventional ubiquitin K6 key and self ubiquitination (Wu-Baer et al., J Biol Chem.2003Sep 12; 278 (37): 34743-6; Chen et al., J Biol Chem.2002Jun 14; 277 (24): 22085-92).Obtainable evidence shows that BRCA1 E3 Ub ligase enzyme activity is that its DNA repairing effect is necessary.Its Ub ligase enzyme activity has been eliminated in the sudden change of tending to cancer in the BRCA1 RING structural domain, and (Ruffner et al., Proc Natl Acad SciU S A.2001Apr 24 to gamma-ray hypersensitivity for the mankind mastopathy cell that these sudden changes can not reverse no BRCA1; 98 (9): 5134-9).In addition, the BRCA1 of siRNA mediation destroys, blocked in the nucleus as accept that DNA in the gamma-rays radiating cell repairs and the nucleus position in activation site, the outpost of the tax office in multipass at deposition (Morris et al., the Hum Mol Genet.2004Apr15 of protein structure; 13 (8): 807-17).The ubiquitin key (K6) that is important to note that the BRCA1 mediation is different from ubiquitin key (K48) (Wu-Baer etal., the J Biol Chem.2003Sep 12 that makes albumen be subjected to the proteasome degraded; 278 (37): 34743-6; Morris et al., HumMol Genet.2004Apr 15; 13 (8): 807-17).Also do not know at present the function of K6 key, but known it can have signal transfer function.
In sum, discovery prompting in these discoveries and the document, the active inhibitor of BRCA1 E3 Ub ligase enzyme may be an effective anticancer agent, because with respect to normal cell (the TP53 positive), it can strengthen the treatment window of DNA disrupting agent to tumour cell (great majority are TP53 feminine genders).Carried out the BRCA1 level to cis-platinum susceptibility enhanced dose-dependently and multipass in the study on deposition of albumen in the nucleus position, whether have cause-effect relationship to observe these incidents.Also studied the chemical inhibitor of BRCA1 E3 Ub ligase enzyme, to establish the effect of multiubiquitination in the DNA destructive is repaired.
Prompting exists other to destroy the evidence of the E3 Ub ligase enzyme that works in the reparation from yeast research (Spence et al., Mol Cell Biol.1995Mar at DNA; 15 (3): 1265-73), show that DNA destroys reparation and need have the specific Ub ligase enzyme of non-proteolytic (K63 key).In order to promote to participate in the evaluation that DNA destroys the ligase enzyme of repairing, we have added the siRNA that has a plurality of E3 ligase enzymes of analog structure domain structure (nameless structural domain ligase enzyme) with BRCA1 in our siRNA library, wish to find to make cell that DNA is destroyed responsive those by our library screening.
The siRNA sequence of Table IV BARD1 and RAD51
Adopted sequence is arranged | Serial ID | The gene title | SEQ?ID?NO | |
5093 | CAGUAAUUCUUAAGGCUAATT | NM_000465 | BARD1 | 1237 |
5094 | CUCCUGAGAAGGUCUGCAATT | NM_000465 | BARD1 | 1238 |
5095 | CGCAGAAGCAGGCUCAACATT | NM_000465 | BARD1 | 1239 |
6920 | GUUAGAGCAGUGUGGCAUATT | NM_002875 | RAD51 | 1240 |
6921 | GGUAUGCACUGCUUAUUGUTT | NM_002875 | RAD51 | 1241 |
6922 | CAGAUUGUAUCUGAGGAAATT | NM_002875 | RAD51 | 1242 |
7. the reference of quoting
All reference of quoting in this is incorporated herein in full for all purposes, its degree is as specific and point out to be introduced as all purposes separately and introduce each independent open source literature or patent or patent application in full.
Can carry out many modifications and variations to the present invention, and not leave its spirit and scope, this it will be apparent to those skilled in the art that.Embodiment described herein only is to illustrate for example, and the present invention only is subjected to the qualification of whole equivalency range of claims and described claim.
Claims (218)
1. the method for identified gene, the product of described gene is regulated the effect of a kind of reagent to a kind of cell of cell type, and described method comprises
(a) a large amount of group of one or more cells of described cell type is contacted with described reagent, wherein the group of each described one or more cell comprises one or more the different siRNAs (siRNA) from a large amount of different siRNA, the gene that described one or more different siRNA target phasings are same, and described a large amount of different siRNA comprise the heterogeneic siRNA in the cell of distinguishing the fixed described cell type of target;
(b) effect to the cell of the described cell type that do not comprise the fixed arbitrary described heterogeneic siRNA of target compares to the effect of the group of each described one or more cell and described reagent with described reagent; With
(c) effect of the cell of the described cell type of arbitrary described heterogeneic siRNA is different calmly to not comprising target if described reagent is to the effect of the group of described one or more cells of described one or more different siRNA of comprising the fixed described gene of target and described reagent, and then identified gene is that described its product is regulated the gene of described reagent to the effect of the cell of described cell type.
2. the process of claim 1 wherein and comprise one or more each described groups of cells among described a large amount of siRNA by before described contact procedure, obtaining with described one or more siRNA transfections.
3. the method for identified gene, the product of described gene is regulated the effect of a kind of reagent to a kind of cell of cell type, and described method comprises
(a) with comprising from a large amount of group of one or more cells of the described cell type of composition transfection of one or more different siRNAs (siRNA) of a large amount of different siRNA each, the gene that described one or more different siRNA target phasings are same, and described a large amount of different siRNA comprise the heterogeneic siRNA in the cell of distinguishing the fixed described cell type of target;
(b) described a large amount of group of one or more cells is contacted with described reagent;
(c) effect to the cell of the described cell type of the fixed arbitrary described heterogeneic siRNA transfection of target of no use compares to the effect of the group of each described one or more cell and described reagent with described reagent; With
(d) effect of the cell of the described cell type of arbitrary described heterogeneic siRNA is different calmly to not comprising target if described reagent is to the effect of the group of described one or more cells of described one or more different siRNA of comprising the fixed described gene of target and described reagent, and then identified gene is that described its product is regulated the gene of described reagent to the effect of the cell of described cell type.
4. any one method of claim 1-3, wherein with described reagent the effect of the cell of the described cell type that do not comprise the fixed arbitrary described heterogeneic siRNA of target is compared, described reagent strengthens the effect of the group of each described one or more cell of comprising described siRNA.
5. any one method of claim 1-3, wherein with described reagent the effect of the cell of the described cell type that do not comprise the fixed arbitrary described heterogeneic siRNA of target is compared, described reagent weakens the effect of the group of each described one or more cell of comprising described siRNA.
6. any one method of claim 1-3, wherein said reagent act on by gene or its encoded protein beyond the fixed arbitrary described different genes of described a large amount of siRNA targets.
7. the method for claim 3, wherein said a large amount of siRNA comprise the different siRNA of the kind of k at least of at least a gene in the fixed described different genes of target, and wherein said k is selected from 2,3,4,5,6 and 10.
8. the method for claim 7, its described one or more different siRNA of described at least a gene that hit surely comprise 2,3,4,5,6 or 10 kind of different siRNA.
9. the method for claim 7, wherein said a large amount of siRNA comprise each the different siRNA of the kind of k at least at least two kinds of different genes in the fixed described different genes of target, and wherein said k is selected from 2,3,4,5,6 and 10.
10. the method for claim 9, described one or more different siRNA of each in its described surely at least two kinds of different genes that hit comprise 2,3,4,5,6 or 10 kind of different siRNA.
11. the method for claim 9, wherein said a large amount of siRNA comprise each the different siRNA of the kind of k at least in the fixed described different genes of target, and wherein said k is selected from 2,3,4,5,6 and 10.
12. the method for claim 11, described one or more different siRNA of each in its described surely different genes that hits comprise 2,3,4,5,6 or 10 kind of different siRNA.
13. the method for claim 5, wherein said cell type are the cancer cells types.
14. the method for claim 13, wherein said cell type are the cancer cells types, and wherein said effect is the growth-inhibiting effect.
15. the method for claim 12, wherein said reagent are the KSP inhibitor.
16. any one method of claim 7-15, wherein said multiple different gene comprises the different gene of N kind at least, wherein N be selected from 5,10,100,1000 with 5000 kinds of different genes.
17. any one method of claim 1-3, wherein said different gene is different native gene.
18. the method for the gene of the primary target gene interaction in the cell of evaluation and a kind of cell type, described method comprises
(a) a large amount of group of one or more cells of described cell type is contacted with a kind of reagent, wherein said reagent is regulated described primary target expression of gene and/or by the proteic activity of described primary target genes encoding, and the group of wherein said cell comprises one or more the different siRNA among a large amount of different siRNA, described one or more different siRNA target phasings gene together, and described a large amount of different siRNA comprise the siRNA of the different secondary genes in the fixed described cell of difference target;
(b) effect to the cell of the described cell type of the siRNA that do not comprise the fixed arbitrary described different secondary genes of target compares to the effect of the group of each described one or more cell and described reagent with described reagent; With
(c) effect of the cell of the described cell type of the siRNA of arbitrary described different secondary genes is different calmly to not comprising target if described reagent is to the effect of the group of described one or more cells of one or more siRNA of comprising the fixed described gene of target and described reagent, and then identified gene is the gene of the described primary target gene interaction in the cell of described and described cell type.
19. the method for claim 18 wherein comprises one or more each described groups of cells among described a large amount of siRNA by obtaining with described one or more siRNA transfections before described contact procedure.
20. the method for the gene of the primary target gene interaction in the cell of evaluation and a kind of cell type, described method comprises
(a) with comprising from a large amount of group of one or more cells of the described cell type of composition transfection of one or more different siRNAs (siRNA) of a large amount of different siRNA each, the gene that described one or more different siRNA target phasings are same, and described a large amount of different siRNA comprise the heterogeneic siRNA in the cell of distinguishing the fixed described cell type of target;
(b) described a large amount of group of one or more cells of described cell type is contacted with a kind of reagent, wherein said reagent is regulated described primary target expression of gene and/or by the proteic activity of described primary target genes encoding;
(c) effect to the cell of the described cell type of the siRNA that do not comprise the fixed arbitrary described different secondary genes of target compares to the effect of the group of each described one or more cell and described reagent with described reagent; With
(d) effect of the cell of the described cell type of the siRNA of arbitrary described different secondary genes is different calmly to not comprising target if described reagent is to the effect of the group of described one or more cells of one or more siRNA of comprising the fixed described gene of target and described reagent, and then identified gene is the gene of the described primary target gene interaction in the cell of described and described cell type.
Decide described primary target gene and make its reticent siRNA 21. any one method of claim 18-20, wherein said reagent are targets.
22. any one method of claim 18-20, described reagent are the inhibitor of described primary target gene.
23. any one method of claim 18-20, wherein with described reagent the effect of the cell of the described cell type of the siRNA that do not comprise the fixed arbitrary described different secondary genes of target is compared, described reagent strengthens the effect of the group of described one or more cells of comprising described one or more siRNA.
24. any one method of claim 18-20, wherein with described reagent the effect of the cell of the described cell type of the siRNA that do not comprise the fixed arbitrary described different secondary genes of target is compared, described reagent weakens the effect of the group of described one or more cells of comprising described one or more siRNA.
25. the method for claim 20, wherein said a large amount of siRNA comprise the different siRNA of the kind of k at least of the fixed secondary gene of at least a described difference of target, and wherein said k is selected from 2,3,4,5,6 and 10.
26. the method for claim 25, its described one or more different siRNA of described at least a gene that hit surely comprise 2,3,4,5,6 or 10 kind of different siRNA.
27. the method for claim 18, wherein said a large amount of siRNA comprise each the different siRNA of the kind of k at least at least two kinds of different genes in the fixed secondary gene of described difference of target, and wherein said k is selected from 2,3,4,5,6 and 10.
28. the method for claim 27, described one or more different siRNA of each in its described surely at least two kinds of different genes that hit comprise 2,3,4,5,6 or 10 kind of different siRNA.
29. the method for claim 27, wherein said a large amount of siRNA comprise each the different siRNA of the kind of k at least in the fixed secondary gene of described difference of target, and wherein said k is selected from 2,3,4,5,6 and 10.
30. the method for claim 29, described one or more different siRNA of each in its described surely different genes that hits comprise 2,3,4,5,6 or 10 kind of different siRNA.
31. the method for claim 22, wherein said primary target gene is KSP.
32. the method for claim 18, wherein said multiple different gene comprises the different gene of N kind at least, wherein N be selected from 5,10,100,1000 with 5000 kinds of different genes.
33. any one method of claim 18-20, wherein said different secondary gene is different native gene.
34. any one method of claim 18-20, wherein said cell type are the cancer cells types.
35. the method for claim 8 or 26, the total siRNA concentration of one or more siRNA described in the wherein said composition is the optimal concentration that is used to make described target gene silence, wherein said optimal concentration is such concentration, when this concentration of further increase, does not increase reticent level substantially.
36. the method for claim 35, wherein said optimal concentration are such concentration, when this concentration of further increase, the increase of reticent level is no more than 20%, be no more than 10% or be no more than 5%.
37. the method for claim 35, the concentration of each of wherein said one or more siRNA is approximately identical.
38. the method for claim 35, wherein said one or more siRNA concentration separately difference each other are less than 50%, less than 20% or less than 10%.
39. the method for claim 35 does not have the concentration of a kind of siRNA to account for more than 80% of total siRNA concentration of described one or more siRNA, more than 50% or more than 20% in the wherein said composition.
40. the method for claim 35, the concentration of at least a siRNA accounts for more than 20% or more than 50% of total siRNA concentration of described one or more siRNA in the wherein said composition.
41. the method for claim 8 or 26, wherein select the concentration of each siRNA in the number of different siRNA and the described composition, make described composition cause any not as the silence of the gene of target less than 10%, less than 1%, less than 0.1% or less than 0.01%.
42. treatment suffers from the mammiferous method of cancer, comprise a kind of reagent to described administration treatment q.s, described reagent is regulated STK6 or TPX2 expression of gene and/or by the proteic activity of described STK6 or TPX2 genes encoding, wherein said Mammals carries out a kind of treatment, and described treatment comprises the KSP inhibitor to described administration treatment q.s.
43. treatment suffers from the mammiferous method of cancer, comprise to described administration i) treatment q.s a kind of reagent, described reagent is regulated STK6 or TPX2 expression of gene and/or by the proteic activity of described STK6 or TPX2 genes encoding with ii) treat the KSP inhibitor of q.s.
44. the method for claim 42 or 43, wherein said reagent reduce STK6 or TPX2 expression of gene described in the cell of described cancer.
45. the method for claim 42 or 43, wherein said reagent comprise the siRNA of fixed described STK6 of target or TPX2 gene.
46. the method for claim 45, wherein said reagent comprise 2,3,4,5,6 or the different siRNA of fixed described STK6 of 10 kind of target or TPX2 gene.
47. the method for claim 46, the total siRNA concentration of different siRNA described in the wherein said reagent is the optimal concentration that is used to make described STK6 or TPX2 gene silencing, wherein said optimal concentration is such concentration, when this concentration of further increase, does not increase reticent level substantially.
48. the method for claim 47, wherein said optimal concentration are such concentration, when this concentration of further increase, the increase of reticent level is no more than 20%, be no more than 10% or be no more than 5%.
49. the method for claim 47, wherein the concentration of each described different siRNA is approximately identical.
50. the method for claim 47, wherein said different siRNA concentration separately difference each other are less than 50%, less than 20% or less than 10%.
51. the method for claim 47 does not have the concentration of a kind of siRNA to account for more than 80% of described total siRNA concentration of described different siRNA, more than 50% or more than 20% in the wherein said reagent.
52. the method for claim 47, the concentration of at least a siRNA accounts for more than 20% or more than 50% of described total siRNA concentration of described different siRNA in the wherein said reagent.
53. the method for claim 47 is wherein selected the concentration of each siRNA in the number of different siRNA and the described reagent, make described reagent cause any not as the silence of the gene of target less than 10%, less than 1%, less than 0.1% or less than 0.01%.
54. the method for claim 45, wherein said Mammals is the people, and wherein said siRNA is selected from the siRNA shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5 and the SEQ ID NO:6.
55. treatment suffers from the mammiferous method of cancer, comprise to described administration i) treatment q.s first kind of reagent, described first kind of reagent is regulated STK6 or TPX2 expression of gene and/or by the proteic activity of described STK6 or TPX2 genes encoding, ii) treat second kind of reagent of q.s, described second kind of reagent is regulated the KSP expression of gene and/or by the proteic activity of described KSP genes encoding.
56. the method for claim 55, wherein said first kind of reagent comprise the siRNA of fixed described TPX2 of target or TPX2 gene, and described second kind of reagent comprises the siRNA of the fixed described KSP gene of target.
57. the method for claim 56, wherein said first kind of reagent comprise 2,3,4,5,6 or the different siRNA of fixed described STK6 of 10 kind of target or TPX2 gene.
58. the method for claim 57, total siRNA concentration of different siRNA is the optimal concentration that is used to make described STK6 or TPX2 gene silencing described in wherein said first kind of reagent, wherein said optimal concentration is such concentration, when this concentration of further increase, does not increase reticent level substantially.
59. the method for claim 58, wherein said optimal concentration are such concentration, when this concentration of further increase, the increase of reticent level is no more than 20%, be no more than 10% or be no more than 5%.
60. the method for claim 58, wherein the concentration of each described different siRNA is approximately identical.
61. the method for claim 58, wherein said different siRNA concentration separately difference each other are less than 50%, less than 20% or less than 10%.
62. the method for claim 58 does not have the concentration of a kind of siRNA to account for more than 80% of described total siRNA concentration of described different siRNA, more than 50% or more than 20% in wherein said first kind of reagent.
63. the method for claim 58, the concentration of at least a siRNA accounts for more than 20% or more than 50% of described total siRNA concentration of described different siRNA in wherein said first kind of reagent.
64. the method for claim 58, wherein select the concentration of each siRNA in the number of different siRNA and the described first kind of reagent, make described first kind of reagent cause any not as the silence of the gene of target less than 10%, less than 1%, less than 0.1% or less than 0.01%.
65. the method for claim 56, wherein said Mammals is the people, and the described siRNA of its described surely STK6 gene that hits is selected from the siRNA shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and the SEQ ID NO:6.
66. the method for claim 45 or 56, wherein said Mammals is the people, and the described siRNA of its described surely TPX2 gene that hits is selected from the siRNA shown in SEQ ID NO:1237, SEQ IDNO:1238 and the SEQ ID NO:1239.
67. the assessment cell is to the method for the Growth Inhibition resistance of KSP inhibitor, described method comprises STK6 or the TPX2 expression of gene level of determining in the described cell, the described expression level that wherein is higher than predetermined threshold levels shows that described cell has resistance to the growth-inhibiting effect of described KSP inhibitor.
68. the method for claim 67, wherein by comprising the method for measuring described STK6 or TPX2 expression of gene level with one or more nucleotide probes, determine described STK6 or TPX2 expression of gene level, each in described one or more polynucleotide probes all comprises the nucleotide sequence in described STK6 or the TPX2 gene.
69. the method for claim 67 or 68, wherein said one or more polynucleotide probes are the polynucleotide probes on microarray.
70. the assessment cell is to the method for the Growth Inhibition resistance of KSP inhibitor, described method comprises the proteic abundance level of determining in the described cell by STK6 or TPX2 genes encoding, the described proteic described abundance level that wherein is higher than predetermined threshold levels shows that described cell has resistance to the growth-inhibiting effect of described KSP inhibitor.
71. the assessment cell is to the method for the Growth Inhibition resistance of KSP inhibitor, described method comprises the proteic activity level of determining in the described cell by STK6 or TPX2 genes encoding, the described activity level that wherein is higher than predetermined threshold levels shows that described cell has resistance to the growth-inhibiting effect of described KSP inhibitor.
72. the method for claim 70 or 71, wherein said cell are people's cells.
73. regulate the method for cell to the Growth Inhibition resistance of KSP inhibitor, comprise a kind of reagent that makes described cells contacting q.s, described reagent is regulated STK6 or TPX2 expression of gene and/or by the proteic activity of described STK6 or TPX2 genes encoding.
74. cell is to the method for the Growth Inhibition resistance of KSP inhibitor in the adjusting Mammals, comprise a kind of reagent to described administration treatment q.s, described reagent is regulated STK6 or TPX2 expression of gene and/or by the proteic activity of described STK6 or TPX2 genes encoding.
75. regulate the method for cell growth, comprise making described cells contacting i) a kind of reagent of q.s, described reagent is regulated STK6 or TPX2 expression of gene and/or by the proteic activity of described STK6 or TPX2 genes encoding and the ii) KSP inhibitor of q.s.
76. claim 73,74 or 75 method, wherein said reagent reduces STK6 described in the described cell or TPX2 expression of gene.
77. claim 73,74 or 75 method, wherein said reagent comprise the siRNA of the fixed described STK6 gene of target.
78. the method for claim 77, wherein said reagent comprise 2,3,4,5,6 or the different siRNA of fixed described STK6 of 10 kind of target or TPX2 gene.
79. the method for claim 78, the total siRNA concentration of different siRNA described in the wherein said reagent is the optimal concentration that is used to make described STK6 or TPX2 gene silencing, wherein said optimal concentration is such concentration, when this concentration of further increase, does not increase reticent level substantially.
80. the method for claim 79, wherein said optimal concentration are such concentration, when this concentration of further increase, the increase of reticent level is no more than 20%, be no more than 10% or be no more than 5%.
81. the method for claim 79, wherein the concentration of each described different siRNA is approximately identical.
82. the method for claim 79, wherein said different siRNA concentration separately difference each other are less than 50%, less than 20% or less than 10%.
83. the method for claim 79 does not have the concentration of a kind of siRNA to account for more than 80% of described total siRNA concentration of described different siRNA, more than 50% or more than 20% in the wherein said reagent.
84. the method for claim 79, the concentration of at least a siRNA accounts for more than 20% or more than 50% of described total siRNA concentration of described different siRNA in the wherein said reagent.
85. the method for claim 79 is wherein selected the concentration of each siRNA in the number of different siRNA and the described reagent, make described reagent cause any not as the silence of the gene of target less than 10%, less than 1%, less than 0.1% or less than 0.01%.
86. the method for claim 77, wherein said cell are people's cells, and wherein said siRNA is selected from the siRNA shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5 and the SEQ ID NO:6.
87. the method for claim 77, wherein said cell are people's cells, and wherein said siRNA is selected from the siRNA shown in SEQ ID NO:1237, SEQ ID NO:1238 and the SEQ ID NO:1239.
88. identify and to regulate the compositions and methods of cell to the Growth Inhibition resistance of KSP inhibitor, wherein said reagent can be regulated STK6 or TPX2 expression of gene and/or by the proteic activity of described STK6 or TPX2 genes encoding, described method comprise described KSP inhibitor when relatively having described reagent to the cell inhibiting effect of expressing described STK6 or TPX2 gene when not having described reagent described KSP inhibitor to expressing the cell inhibiting effect of described STK6 or TPX2 gene, the described inhibiting difference of wherein said KSP inhibitor identifies that described reagent can regulate the Growth Inhibition resistance of described cell to the KSP inhibitor.
89. identify and can regulate the compositions and methods of cell that wherein said reagent can regulate STK6 or TPX2 expression of gene and/or by the proteic activity of described STK6 or TPX2 genes encoding, described method comprises to the Growth Inhibition resistance of KSP inhibitor:
(a) first kind of cell of expressing described STK6 or TPX2 gene contacted existing with described KSP inhibitor, and measure first kind of growth-inhibiting effect;
(b) second kind of cell of expressing described STK6 or TPX2 gene contacted with described KSP inhibitor, and measure second kind of growth-inhibiting effect; With
(c) compare in described step (a) and described first kind and second kind of restraining effect of measuring (b),
Difference between wherein said first kind and the second kind of restraining effect identifies that described reagent can regulate the Growth Inhibition resistance of cell to the KSP inhibitor.
90. the method for claim 88 or 89, wherein said reagent comprise the molecule that reduces described STK6 or TPX2 genetic expression.
91. the method for claim 88 or 89, wherein said reagent are the siRNA that target is decided described STK6 or TPX2 gene.
92. the method for claim 91, wherein said cell are people's cells, and wherein said siRNA is selected from the siRNA shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5 and the SEQ ID NO:6.
93. the method for claim 91, wherein said cell are people's cells, and wherein said siRNA is selected from the siRNA shown in SEQ ID NO:1237, SEQ ID NO:1238 and the SEQ ID NO:1239.
94. comprise the cell of one or more different siRNAs (siRNA), STK6 or TPX2 gene in the fixed described cell of described siRNA target.
95. the cell of claim 94, wherein said one or more different siRNA comprise 2,3,4,5,6 or 10 kind of different siRNA.
96. the cell of claim 95, wherein said cell is to produce by the composition transfection with described one or more different siRNA, total siRNA concentration of wherein said composition is the optimal concentration that is used to make described STK6 or TPX2 gene silencing, wherein said optimal concentration is such concentration, when this concentration of further increase, do not increase reticent level substantially.
97. the cell of claim 96, wherein said optimal concentration are such concentration, when this concentration of further increase, the increase of reticent level is no more than 20%, be no more than 10% or be no more than 5%.
98. the cell of claim 96, wherein the concentration of each described different siRNA is approximately identical.
99. the cell of claim 96, wherein said different siRNA concentration separately difference each other are less than 50%, less than 20% or less than 10%.
100. the cell of claim 96, do not have in the wherein said composition concentration of a kind of siRNA account for more than 80% of total siRNA concentration of described different siRNA, more than 50% or more than 20%.
101. the cell of claim 96, the concentration of at least a siRNA accounts for more than 20% or more than 50% of described total siRNA concentration of described different siRNA in the wherein said composition.
102. the cell of claim 96, wherein select the concentration of each siRNA in the number of different siRNA and the described composition, make described composition cause any not as the silence of the gene of target less than 10%, less than 1%, less than 0.1% or less than 0.01%.
103. the cell of claim 94, wherein said cell are people's cells.
104. the cell of claim 103, wherein said cell is people's cell, and each of wherein said one or more different siRNA is selected from the siRNA shown in SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and the SEQ IDNO:6.
105. the cell of claim 103, wherein said cell are people's cells, and wherein said siRNA is selected from the siRNA shown in SEQ ID NO:1237, SEQ ID NO:1238 and the SEQ ID NO:1239.
106. the cell of claim 94, wherein said cell are the mouse cells.
107. be used for the microarray of diagnosis cell to the Growth Inhibition resistance of KSP inhibitor, described microarray comprises one or more polynucleotide probes, wherein each described polynucleotide probes comprises the nucleotide sequence in STK6 or the TPX2 gene.
108. be used for the test kit of diagnosis cell to the Growth Inhibition resistance of KSP inhibitor, described test kit comprises one or more polynucleotide probes that place one or more containers, and wherein each described polynucleotide probes comprises the nucleotide sequence in STK6 or the TPX2 gene.
109. be used to screen a kind of test kit of reagent, described reagent is regulated the Growth Inhibition resistance of cell to the KSP inhibitor, described test kit comprises the cell of (i) claim 94 that places one or more containers; (ii) KSP inhibitor.
110. be used for the treatment of the mammiferous test kit of suffering from cancer, this test kit comprises the adjusting STK6 of (i) q.s that places one or more containers or TPX2 expression of gene and/or by the proteic active reagent of described STK6 or TPX2 genes encoding; (ii) KSP inhibitor.
111. any one method of claim 42-43,67,70-71,74-75 and 88-89, wherein said KSP inhibitor are (1S)-1-{[(2S)-4-(2, the 5-difluorophenyl)-2-phenyl-2,5-dihydro-1H-pyrroles-1-yl] carbonyl }-2-methyl propylamine.
112. claim 1,2 or 3 method, wherein the component to each described one or more cell drives capable contact procedure (a) into.
113. claim 18,19 or 20 method, wherein the component to each described one or more cell drives capable contact procedure (a) into.
114. the test kit of claim 109 or 110, wherein said KSP inhibitor are (1S)-1-{[(2S)-4-(2, the 5-difluorophenyl)-2-phenyl-2,5-dihydro-1H-pyrroles-1-yl] carbonyl }-2-methyl propylamine.
115. the method for the gene of the primary target gene interaction in the cell of evaluation and a kind of cell type, described method comprises
(a) make a kind of reagent of one or more cells contacting of described cell type, wherein said reagent is regulated the secondary target expression of gene and/or by the proteic activity of described secondary target genes encoding, and first siRNA (siRNA) of the fixed described primary target gene of wherein said one or more cell expressing targets;
(b) more described reagent is to the effect to the cell of the described cell type of not expressing a described siRNA of the effect of described one or more cells of described clone and described reagent; With
(c) if described reagent is different to the effect of the cell of the described cell type of not expressing a described siRNA to the effect of described one or more cells of expressing a described siRNA and described reagent, then identify described secondary target gene for the cell of described cell type in the gene of primary target gene interaction.
116. the method for the gene of the primary target gene interaction in the cell of evaluation and a kind of cell type, described method comprises
(a) clone of the cell of the described cell type of first siRNA (siRNA) of the fixed described primary target gene of generation expression target;
(b) make a kind of reagent of one or more cells contacting of described clone, wherein said reagent is regulated the secondary target expression of gene and/or by the proteic activity of described secondary target genes encoding;
(c) more described reagent is to the effect to the cell of the described cell type of not expressing a described siRNA of the effect of described one or more cells of described clone and described reagent; With
(d) if described reagent is different to the effect of the cell of the described cell type of not expressing a described siRNA to the effect of described one or more cells of expressing a described siRNA and described reagent, then identify described secondary target gene for the cell of described cell type in the gene of primary target gene interaction.
117. the method for claim 116, a wherein said siRNA is expressed by the nucleotide sequence that is incorporated in the described cellular genome.
118. the method for claim 116, wherein said reagent comprise the fixed described secondary target gene of target and make one or more the 2nd siRNA of its silence.
119. the method for claim 116, wherein said reagent are the inhibitor of described secondary target gene.
120. the method for claim 118 is wherein compared the effect of the cell of the described cell type of not expressing a described siRNA with described reagent, described reagent strengthens the effect of described one or more cells of expressing a described siRNA.
121. the method for claim 118 is wherein compared the effect of the cell of the described cell type of not expressing a described siRNA with described reagent, described reagent weakens the effect of described one or more cells of expressing a described siRNA.
122. the method for claim 120, wherein said one or more the 2nd siRNA comprise the different siRNA of k kind at least, and wherein said k is selected from 2,3,4,5,6 and 10.
123. the method for claim 122, total siRNA concentration of the siRNA that the k kind is different at least described in the wherein said reagent is the optimal concentration that is used to make described secondary target gene silencing, wherein said optimal concentration is such concentration, when this concentration of further increase, do not increase reticent level substantially.
124. the method for claim 123, wherein said optimal concentration are such concentration, when this concentration of further increase, the increase of reticent level is no more than 20%, be no more than 10% or be no more than 5%.
125. the method for claim 123, the concentration of each of the siRNA that the wherein said kind of k at least is different is approximately identical.
126. the method for claim 123, the different siRNA concentration separately difference each other of the wherein said kind of k at least are less than 50%, less than 20% or less than 10%.
127. the method for claim 123 does not have the concentration of a kind of siRNA to account for more than 80% of described total siRNA concentration of described different siRNA, more than 50% or more than 20% in the wherein said reagent.
128. the method for claim 123, the concentration of at least a siRNA accounts for more than 20% or more than 50% of described total siRNA concentration of the different siRNA of the described kind of k at least in the wherein said reagent.
129. the method for claim 123 is wherein selected the concentration of each siRNA in the number of different siRNA and the described reagent, make described reagent cause any not as the silence of the gene of target less than 10%, less than 1%, less than 0.1% or less than 0.01%.
130. the method for claim 122, wherein said cell type are the cancer cells types, and wherein said primary target gene is p53.
131. the method for claim 130 further comprises step
(e) in a large amount of different secondary target genes each, repeating step (b)-(d).
132. the method for claim 131, wherein said a large amount of secondary target gene comprise the different genes of number that is selected from down group at least: 5,10,100,1000 with 5000 kinds of different genes.
133. the method for claim 132, wherein said effect are the change of the cell of described cell type to the susceptibility of medicine.
134. the method for claim 133, wherein said medicine are the DNA disrupting agents.
135. the method for claim 134, wherein said DNA disrupting agent is selected from topoisomerase I inhibitor, topoisomerase II inhibitor, DNA wedding agent and ionizing rays.
136. the method for claim 135, wherein said DNA disrupting agent is selected from Zorubicin, camptothecine and cis-platinum.
137. assess the reactive method of a kind of cell of cell type, comprise to pharmacological agent
(a) make the described medicine of one or more cells contacting of described cell type, wherein said one or more cell expressing targets are decided first siRNA (siRNA) of primary target gene, and wherein said one or more cells are accepted a kind of processing of composition, and described composition is regulated one or more secondary target expression of gene and/or respectively by the proteic activity of described one or more secondary target genes encodings;
(b) make the described medicine of one or more cells contacting of described cell type, wherein said one or more cells are not expressed the siRNA (siRNA) of the fixed described primary target gene of target, and wherein said one or more cells are accepted the governing stage target gene expression and/or by the processing of the proteic active described reagent of described secondary target genes encoding; With
(c) the described medicine of relatively measuring in step (a) is to the effect to described one or more cells of the described medicine of the effect of described one or more cells and measurement in step (b), thereby assesses the reactivity of described cell to described pharmacological agent.
138. assess the reactive method of a kind of cell of cell type to pharmacological agent, described method comprises
(a) clone of cell of described subclass type that target is decided first siRNA (siRNA) of primary target gene is expressed in preparation;
(b) make the described clone's who expresses a described siRNA the described medicine of one or more cells contacting, wherein said one or more cells are accepted the governing stage target gene expression and/or by the processing of the proteic active reagent of described secondary target genes encoding;
(c) make the described medicine of one or more cells contacting of the described cell type of the siRNA (siRNA) of not expressing the fixed described primary target gene of target, wherein said one or more cells are accepted the governing stage target gene expression and/or by the processing of the proteic active described reagent of described secondary target genes encoding; With
(d) the described medicine of relatively measuring in step (b) is to the effect to described one or more cells of the described medicine of the effect of described one or more cells and measurement in step (c), thereby assesses the reactivity of described cell to described pharmacological agent.
139. the method for claim 137 or 138 is wherein compared the effect of the cell of the described cell type of not expressing a described siRNA with described medicine, described medicine strengthens the effect of one or more cells of expressing a described siRNA.
140. the method for claim 137 or 138 is wherein compared the effect of the cell of the described cell type of not expressing a described siRNA with described medicine, described medicine weakens the effect of one or more cells of expressing a described siRNA.
141. the method for claim 137 or 138, wherein said composition comprise one or more inhibitor of described one or more secondary target genes.
142. the method for claim 137 or 138, wherein said composition comprise fixed described one or more second target genes of target and make one or more the 2nd siRNA of its silence.
143. the method for claim 142, wherein said one or more the 2nd siRNA comprise the different siRNA of k kind at least, and wherein said k is selected from 2,3,4,5,6 and 10.
144. the method for claim 143, total siRNA concentration of the siRNA that the k kind is different at least described in the wherein said reagent is the optimal concentration that is used to make described secondary target gene silencing, wherein said optimal concentration is such concentration, when this concentration of further increase, do not increase reticent level substantially.
145. the method for claim 144, wherein said optimal concentration are such concentration, when this concentration of further increase, the increase of reticent level is no more than 20%, be no more than 10% or be no more than 5%.
146. the method for claim 144, the concentration of each of the siRNA that the wherein said kind of k at least is different is approximately identical.
147. the method for claim 144, the different siRNA concentration separately difference each other of the wherein said kind of k at least are less than 50%, less than 20% or less than 10%.
148. the method for claim 144 does not have the concentration of a kind of siRNA to account for more than 80% of described total siRNA concentration of described different siRNA, more than 50% or more than 20% in the wherein said reagent.
149. the method for claim 144, the concentration of at least a siRNA accounts for more than 20% or more than 50% of described total siRNA concentration of the different siRNA of the described kind of k at least in the wherein said reagent.
150. the method for claim 144 is wherein selected the concentration of each siRNA in the number of different siRNA and the described reagent, make described reagent cause any not as the silence of the gene of target less than 10%, less than 1%, less than 0.1% or less than 0.01%.
151. the method for claim 137 or 138, wherein said cell type are the cancer cells types, and wherein said primary target gene is p53.
152. the method for claim 138 further comprises step
(e) in a large amount of different secondary target genes each, repeating step (b)-(d).
153. the method for claim 137 further comprises step
(e) in a large amount of different secondary target genes each, repeating step (a)-(b).
154. the method for claim 152 or 153, wherein said a large amount of secondary target gene comprise the different genes of number that is selected from down group at least: 5,10,100,1000 with 5000 kinds of different genes.
155. the method for claim 154, wherein said medicine are the DNA disrupting agents.
156. the method for claim 155, wherein said DNA disrupting agent is selected from topoisomerase I inhibitor, topoisomerase II inhibitor, DNA wedding agent and ionizing rays.
157. the method for claim 156, wherein said DNA disrupting agent is selected from Zorubicin, camptothecine and cis-platinum.
158. treatment suffers from the mammiferous method of cancer, comprise a kind of reagent to described administration treatment q.s, described reagent is regulated a kind of expression of gene and/or by the proteic activity of described genes encoding, wherein said Mammals carries out a kind of treatment, and described treatment comprises the composition that comprises one or more DNA disrupting agents to described administration treatment q.s.
159. treatment suffers from the mammiferous method of cancer, comprise to described administration i) treatment q.s a kind of reagent, described reagent is regulated a kind of expression of gene and/or by the proteic activity of described genes encoding with ii) treat the composition that comprises one or more DNA disrupting agents of q.s.
160. the method for claim 158 or 159, wherein said reagent make the expression decreased of described gene in the cell of described cancer.
161. the method for claim 158 or 159, wherein said reagent strengthen the expression of described gene in the cell of described cancer.
162. the method for claim 161, wherein said one or more DNA disrupting agents are selected from topoisomerase I inhibitor, topoisomerase II inhibitor, DNA wedding agent and ionizing rays, and wherein said gene is selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51.
163. the method for claim 161, wherein said one or more DNA disrupting agents are selected from Zorubicin, camptothecine and cis-platinum, and wherein said gene is selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51.
164. the method for claim 163, wherein said reagent comprise the siRNA of the fixed described gene of target.
165. the assessment cell is to a kind of method of Growth Inhibition susceptibility of reagent, described method comprises each transcriptional level of one or more genes of determining in the described cell, wherein be lower than each described transcriptional level of each gene of predetermined threshold levels, show that described cell has susceptibility to the growth-inhibiting effect of described DNA disrupting agent.
166. the method for claim 165, wherein said reagent is the DNA disrupting agent, it is selected from topoisomerase I inhibitor, topoisomerase II inhibitor, DNA wedding agent and ionizing rays, and wherein said gene is selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51.
167. the method for claim 165, wherein said DNA disrupting agent is selected from Zorubicin, camptothecine and cis-platinum, and wherein said gene is selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51.
168. any one method of claim 166-167, wherein said one or more genes comprise about at least 5 to about 50 kinds of different genes.
169. the method for claim 168, wherein each described transcriptional level is than low 1.5 times, 2 times or 3 times of described threshold levels.
170. any one method of claim 166-167, wherein by comprising the method for measuring described gene transcription level with one or more nucleotide probes, determine described gene transcription level, each in described one or more polynucleotide probes all comprises the nucleotide sequence in the described gene.
171. the method for claim 170, wherein said one or more polynucleotide probes are the polynucleotide probes on microarray.
172. be used to assess the method for cell to the Growth Inhibition susceptibility of DNA disrupting agent, described method comprises the proteic abundance level of determining in the described cell by a kind of genes encoding, wherein be lower than the described proteic described abundance level of predetermined threshold levels, show that described cell has susceptibility to the growth-inhibiting effect of described DNA disrupting agent.
173. the assessment cell is to the method for the Growth Inhibition susceptibility of DNA disrupting agent, described method comprises the proteic activity level of determining in the described cell by described genes encoding, the described activity level that wherein is higher than predetermined threshold levels shows that described cell has susceptibility to the growth-inhibiting effect of described DNA disrupting agent.
174. the method for claim 172 or 173, wherein said DNA disrupting agent is selected from topoisomerase I inhibitor, topoisomerase II inhibitor, DNA wedding agent and ionizing rays, and wherein said gene is selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51.
175. the method for claim 174, wherein said DNA disrupting agent is selected from Zorubicin, camptothecine and cis-platinum, and wherein said gene is selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51.
176. the method for claim 172 or 173, wherein said cell are people's cells.
177. regulate the method for cell to DNA destructive susceptibility, described method comprises makes described cell contact with a kind of reagent of q.s, and described reagent is regulated the expression of gene that is selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51 and/or by the proteic activity of described genes encoding.
178. the method for claim 177, wherein said DNA destroys and is caused by the DNA disrupting agent.
179. the method for claim 178, wherein said DNA disrupting agent is selected from topoisomerase I inhibitor, topoisomerase II inhibitor, DNA wedding agent and ionizing rays.
180. the method for claim 179, wherein said DNA disrupting agent is selected from Zorubicin, camptothecine and cis-platinum.
181. regulate the method for cell growth, comprise and make described cells contacting i) a kind of reagent of q.s, described reagent is regulated the expression of gene that is selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51 and/or by the proteic activity of described genes encoding; The ii) DNA disrupting agent of q.s.
182. the method for claim 177 or 181, wherein said reagent reduces expression of gene described in the described cell.
183. the method for claim 177 or 181, wherein said reagent comprise the siRNA of the fixed described gene of target.
184. the method for claim 183, wherein said reagent comprise 2,3,4,5,6 or 10 kind of different siRNA of the fixed described gene of target.
185. the method for claim 184, total siRNA concentration of different siRNA is the optimal concentration that is used to make described gene silencing described in the wherein said reagent, wherein said optimal concentration is such concentration, when this concentration of further increase, does not increase reticent level substantially.
186. the method for claim 185, wherein said optimal concentration are such concentration, when this concentration of further increase, the increase of reticent level is no more than 20%, be no more than 10% or be no more than 5%.
187. the method for claim 185, wherein the concentration of each described different siRNA is approximately identical.
188. the method for claim 185, wherein said different siRNA concentration separately difference each other are less than 50%, less than 20% or less than 10%.
189. the method for claim 185 does not have the concentration of a kind of siRNA to account for more than 80% of described total siRNA concentration of described different siRNA, more than 50% or more than 20% in the wherein said reagent.
190. the method for claim 185, the concentration of at least a siRNA accounts for more than 20% or more than 50% of described total siRNA concentration of described different siRNA in the wherein said reagent.
191. the method for claim 185 is wherein selected the concentration of each siRNA in the number of different siRNA and the described reagent, make described reagent cause any not as the silence of the gene of target less than 10%, less than 1%, less than 0.1% or less than 0.01%.
192. identify and to regulate the compositions and methods of cell to the Growth Inhibition susceptibility of DNA disrupting agent, wherein said reagent can be regulated and be selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, the expression of gene of BARD1 and RAD51 and/or by the proteic activity of described genes encoding, this method comprise described DNA disrupting agent when relatively having described reagent to the cell inhibiting effect of expressing said gene when not having described reagent described DNA disrupting agent to the cell inhibiting effect of expressing said gene, the described inhibiting difference of wherein said DNA disrupting agent identifies that described reagent can regulate the Growth Inhibition susceptibility of described cell to the DNA disrupting agent.
193. identify and to regulate the compositions and methods of cell to the Growth Inhibition susceptibility of DNA disrupting agent, wherein said reagent can regulate the expression of gene that is selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51 and/or by the proteic activity of described genes encoding, described method comprises
(a) exist the cell that makes first kind of expressing said gene under the condition of described reagent to contact, and measuring first kind of growth-inhibiting effect with described DNA disrupting agent;
(b) cell of second kind of expressing said gene is contacted with described DNA disrupting agent, and measure second kind of growth-inhibiting effect;
(c) compare in described step (a) and described first kind and second kind of restraining effect of measuring (b),
Difference between wherein said first kind and the second kind of restraining effect identifies that described reagent can regulate the Growth Inhibition susceptibility of described cell to described DNA disrupting agent.
194. the method for claim 192 or 193, wherein said cell expressing target is decided the siRNA of primary target gene.
195. the method for claim 194, wherein said primary target gene is p53.
196. the method for claim 192 or 193, wherein said reagent comprises the molecule that reduces described genetic expression.
197. the method for claim 196, wherein said reagent comprise the siRNA of the fixed described gene of target.
198. the method for claim 197, wherein said reagent comprise 2,3,4,5,6 or 10 kind of different siRNA of the fixed described gene of target.
199. the method for claim 198, total siRNA concentration of different siRNA is the optimal concentration that is used to make described gene silencing described in the wherein said reagent, wherein said optimal concentration is such concentration, when this concentration of further increase, does not increase reticent level substantially.
200. the method for claim 199, wherein said optimal concentration are such concentration, when this concentration of further increase, the increase of reticent level is no more than 20%, be no more than 10% or be no more than 5%.
201. the method for claim 199, wherein the concentration of each described different siRNA is approximately identical.
202. the method for claim 199, wherein said different siRNA concentration separately difference each other are less than 50%, less than 20% or less than 10%.
203. the method for claim 199 does not have the concentration of a kind of siRNA to account for more than 80% of described total siRNA concentration of described different siRNA, more than 50% or more than 20% in the wherein said reagent.
204. the method for claim 199, the concentration of at least a siRNA accounts for more than 20% or more than 50% of described total siRNA concentration of described different siRNA in the wherein said reagent.
205. the method for claim 199 is wherein selected the concentration of each siRNA in the number of different siRNA and the described reagent, make described reagent cause any not as the silence of the gene of target less than 10%, less than 1%, less than 0.1% or less than 0.01%.
206. comprise the cell of one or more different siRNAs (siRNA), the gene that is selected from EPHB3, WEE1, ELK1, BRCA1, BRCA2, BARD1 and RAD51 in the fixed described cell of described siRNA target.
207. the method for claim 206, wherein said one or more siRNA comprise 2,3,4,5,6 or 10 kind of different siRNA.
208. the method for claim 206, wherein said cell are people's cells.
209. the method for claim 208, wherein said cell are the mouse cells.
210. be used for the microarray of diagnosis cell to the Growth Inhibition susceptibility of DNA disrupting agent, described microarray comprises one or more polynucleotide probes, and wherein each described polynucleotide probes comprises the nucleotide sequence in one or more genes that are selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51.
211. be used for the test kit of diagnosis cell to the Growth Inhibition susceptibility of DNA disrupting agent, described test kit comprises one or more polynucleotide probes that place one or more containers, and wherein each described polynucleotide probes comprises the nucleotide sequence in the gene that is selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51.
Regulate the test kit of cell to the reagent of the Growth Inhibition susceptibility of DNA disrupting agent 212. be used to screen, described test kit comprises any one cell of (i) the claim 206-211 that places one or more containers; (ii) described DNA disrupting agent.
213. be used for the treatment of the mammiferous test kit of suffering from cancer, described test kit comprises a kind of reagent of (i) q.s that places one or more containers, and described reagent is regulated the expression of gene that is selected from EPHB3, WEE1, ELK1, STK6, BRCA1, BRCA2, BARD1 and RAD51 and/or by the proteic activity of described genes encoding; (ii) DNA disrupting agent.
214. any one method of claim 192-193, wherein said DNA disrupting agent is selected from topoisomerase I inhibitor, topoisomerase II inhibitor, DNA wedding agent and ionizing rays.
215. the method for claim 214, wherein said DNA disrupting agent is selected from Zorubicin, camptothecine and cis-platinum.
216. the test kit of claim 212, wherein said DNA disrupting agent is selected from topoisomerase I inhibitor, topoisomerase II inhibitor, DNA wedding agent and ionizing rays.
217. the test kit of claim 216, wherein said DNA disrupting agent is selected from Zorubicin, camptothecine and cis-platinum.
218. claim 21,117,137 or 138 method are wherein controlled the reticent level of described primary target gene.
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